Frequently Asked Questions

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Categories

Applications
  1. Array-Based Methylation Analysis
  2. Array-Based Transcriptome Analysis
  3. Custom Genotyping
  4. Custom Low- to Mid-plex Gene Expression Analysis
  5. Custom Low- to Mid-plex Genotyping
  6. Custom Low- to Mid-plex Methylation Analysis
  7. Cytogenetic Analysis
  8. DNA Sequencing
  9. DNA-Protein Interaction Analysis (ChIP-Seq)
  10. FFPE Sample Analysis
  11. Focused Genotyping
  12. Gene Regulation Analysis
  13. Linkage Analysis
  14. Protein Screening
  15. Real-Time PCR
  16. SNP Discovery and Structural Variation Analysis
  17. SNP Genotyping and CNV Analysis
  18. Sequencing
  19. Sequencing-Based Methylation Analysis
  20. Sequencing-Based Transcriptome Analysis
  21. Small RNA Discovery and Analysis
  22. Whole-Genome Gene Expression Analysis
  23. Whole-Genome Genotyping and Copy Number Analysis
Assays
  1. Chromatin Immunoprecipitation Sequencing (ChIP-Seq)
  2. Direct Hybridization Assay
  3. GT
  4. GoldenGate Genotyping Assay
  5. GoldenGate Methylation Assay
  6. Infinium DNA Analysis Assay
  7. Infinium Methylation Assay
  8. MSI
  9. Mate-Pair Library Sequencing
  10. Multiplex Sequencing
  11. Nextera Enrichment
  12. Nextera Rapid Capture
  13. Nextera Sample Prep
  14. Nextera XT Sample Prep
  15. NuPCR
  16. Paired-End Sequencing
  17. Single-Read Sequencing
  18. Small RNA Discovery and Analysis
  19. TruSeq ChIP Sample Prep
  20. TruSeq Custom Amplicon Library Prep
  21. TruSeq DNA PCR-Free Sample Prep
  22. TruSeq DNA Sample Prep
  23. TruSeq DNA Sample Prep v1
  24. TruSeq Enrichment
  25. TruSeq Nano DNA Sample Prep
  26. TruSeq RNA Access Library Prep
  27. TruSeq RNA Sample Prep
  28. TruSeq RNA Sample Prep v2
  29. TruSeq Sample Prep
  30. TruSeq Small RNA Sample Prep
  31. TruSeq Stranded Total RNA Sample Prep
  32. TruSeq Stranded mRNA Sample Prep
  33. TruSeq Synthetic Long-Read DNA Library Prep
  34. TruSeq Targeted RNA Expression
  35. TruSight One Sample Prep
  36. VeraCode GoldenGate Genotyping Assay
  37. VeraCode GoldenGate Methylation Assay
  38. VeraCode Protein Assay
  39. Whole-Genome DASL HT Assay
  40. mRNA-Seq
  41. miRNA Expression Profiling Assay
Asset Type
  1. FAQ
Epigenetics
  1. Array-Based Methylation Analysis
  2. ChIP-Seq
  3. Sequencing-Based Methylation Analysis
  4. Small RNA Analysis
FAQ Category
  1. Analysis
  2. Assay Design
  3. Cluster Generation
  4. Compatibility
  5. Errors
  6. File Formats
  7. Gene Expression
  8. General
  9. Genotyping
  10. Input
  11. Libraries
  12. Library Evaluation
  13. Performance
  14. Polyploidy
  15. Primers
  16. Protocol
  17. Reagent Kits
  18. Reagents
  19. Run Setup
  20. Sequencing
  21. Software
  22. Uncategorized
  23. Workflow
Products
  1. African American Admixture Panel
  2. Bovine3K
  3. BovineHD
  4. BovineSNP50 v2 Whole-Genome Genotyping
  5. CanineHD Whole-Genome Genotyping
  6. ChIP-Seq DNA Sample Prep Kit
  7. Epigenetic and Gene Regulation Analysis
  8. Genomic DNA Sample Prep Kit
  9. Genomic DNA Sample Prep Oligo Only Kit
  10. GoldenGate Cancer SNP Panel
  11. GoldenGate DNA Test Panel
  12. GoldenGate Human V Panel Set
  13. GoldenGate Indexing Kit v1
  14. GoldenGate Indexing Universal-32 v8
  15. GoldenGate MHC Exon-Centric Panel
  16. GoldenGate MHC Mapping Panel
  17. GoldenGate MHC Panel Set
  18. GoldenGate Mouse LD Linkage
  19. GoldenGate Mouse MD Linkage
  20. GoldenGate Universal-12 BeadChip
  21. GoldenGate Universal-32 BeadChip
  22. GoldenGate Universal-XT BeadChip
  23. HiSeq SBS Kit v4 - HS
  24. Human Linkage-24
  25. Human1KGP-12 BeadChip
  26. Human1M-Duo DNA Analysis
  27. Human1M-Duo+ DNA Analysis
  28. Human610-Quad
  29. Human660W-Quad v1 DNA Analysis
  30. HumanCNV100W
  31. HumanCytoSNP-12 DNA Analysis
  32. HumanHT-12 v4 Expression BeadChip Kit
  33. HumanLinkage V Panel Set
  34. HumanLinkage-12 Panels
  35. HumanOmni1-Quad
  36. HumanOmni1S-8
  37. HumanOmni2.5-Quad
  38. HumanOmniExpress
  39. HumanOmniExpress+
  40. Infinium HumanExome BeadChip
  41. Infinium HumanExome+ BeadChip
  42. Infinium HumanMethylation27 BeadChip Kit
  43. Infinium HumanMethylation450 BeadChip Kit
  44. Infinium HumanOmni5Exome
  45. Infinium HumanOmni5Exome+
  46. Infinium HumanOmniExpressExome
  47. Infinium HumanOmniExpressExome+
  48. Infinium Methylation450k FFPE BeadChip Kit
  49. Infinium Oncoarray-500K BeadChip
  50. MaizeSNP50 Whole-Genome Genotyping
  51. Mate Pair Library Prep Kit
  52. Mate Pair Library Prep Kit v2
  53. MiSeq Reagent Kit
  54. MouseRef-8 v2 Expression BeadChip Kit
  55. MouseWG-6 v2 Expression BeadChip Kit
  56. Multiplexing Sample Preparation Oligonucleotide Kit
  57. NextSeq 500 Kit
  58. Nextera DNA Sample Prep Kit
  59. Nextera Enrichment Kit
  60. Nextera Mate Pair Kit
  61. Nextera Rapid Capture Custom Kit
  62. Nextera Rapid Capture Exome Kit
  63. Nextera XT Sample Prep Kit
  64. NuPCR
  65. OvineSNP50 Whole-Genome Genotyping
  66. Paired-End DNA Sample Prep Kit v1
  67. PhiX Control
  68. PorcineSNP60 Whole-Genome Genotyping
  69. RatRef-12 Expression BeadChip Kit
  70. Real-Time PCR
  71. Sequencing - DNA Sequencing
  72. Sequencing - RNA Sequencing
  73. Small RNA Oligo Only Sample Prep Kit
  74. Small RNA Sample Prep Kit
  75. TruSeq Amplicon - Cancer Panel Library Prep Kit
  76. TruSeq ChIP Sample Prep Kit
  77. TruSeq Custom Amplicon Kit
  78. TruSeq Custom Enrichment
  79. TruSeq DNA HT Sample Prep Kit
  80. TruSeq DNA LT Sample Prep Kit
  81. TruSeq DNA PCR-Free HT Sample Prep Kit
  82. TruSeq DNA PCR-Free LT Sample Prep Kit
  83. TruSeq DNA Sample Prep Kit v1
  84. TruSeq Dual Index Sequencing Primers Kit
  85. TruSeq Enrichment Kits
  86. TruSeq Exome Enrichment
  87. TruSeq Nano DNA HT Sample Prep Kit
  88. TruSeq Nano DNA LT Sample Prep Kit
  89. TruSeq Paired-End Cluster Kit v3 - cBot - HS
  90. TruSeq RNA Access Library Prep Kit
  91. TruSeq RNA Sample Prep Kit
  92. TruSeq RNA Sample Prep Kit v2
  93. TruSeq SBS Kits
  94. TruSeq Sample Prep Kits
  95. TruSeq Single-Read Cluster Kit v3 - cBot - HS
  96. TruSeq Small RNA Sample Prep Kit
  97. TruSeq Stranded Total RNA HT Sample Prep Kit
  98. TruSeq Stranded Total RNA LT Sample Prep Kit
  99. TruSeq Stranded mRNA HT Sample Prep Kit
  100. TruSeq Stranded mRNA LT Sample Prep Kit
  101. TruSeq Synthetic Long-Read DNA Library Prep Kit
  102. TruSeq Targeted RNA Expression Kit
  103. TruSight DNA Amplicon Sequencing Panel Kit
  104. TruSight One Sample Prep Kit
  105. TruSight Sample Prep Kit
  106. VeraCode GoldenGate Methylation Analysis Kit
  107. Whole Genome DASL HT Assay Kits
  108. cBot
  109. cDNA Synthesis Kit
  110. iSelect 12x1 HD
  111. iSelect 24x1 HD
  112. iSelect 24x1 HTS
  113. iSelect Custom Genotyping
  114. mRNA-Seq 8-Sample Prep Kit
Services
  1. Genotyping
Software
  1. Assay Design Tool (ADT)
  2. BaseSpace
  3. Bisulfite Sequencing Analysis
  4. CASAVA
  5. Decode File Client
  6. DesignStudio
  7. Eco Control Software (ECS)
  8. Experiment Manager
  9. GenomeStudio
  10. HiSeq Analysis Software
  11. HiSeq Control Software (HCS)
  12. KaryoStudio
  13. MiSeq Control Software (MCS)
  14. MiSeq Reporter
  15. NextSeq Control Software(NCS)
  16. OLB
  17. RTA
  18. Sequencer System Software
  19. Sequencing Analysis Viewer (SAV)
  20. Sequencing Control Software (SCS)
  21. VariantStudio
  22. iControlDB
Systems
  1. BeadXpress Reader
  2. Cluster Station
  3. Eco Real-Time PCR System
  4. Genome Analyzer IIe
  5. Genome Analyzer IIx
  6. HiScanSQ
  7. HiSeq 1000
  8. HiSeq 1500
  9. HiSeq 2000
  10. HiSeq 2500
  11. HiSeq X
  12. MiSeq
  13. NextSeq 500 Desktop Genome Sequencer
  14. cBot
Technologies
  1. BeadArray
  2. General Questions
  3. Illumina Array
  4. Illumina Sequencing
  5. Real-Time PCR
Workflow
  1. Analysis Software
  2. Cluster Generation
  3. Experimental Design
  4. General Information
  5. Instrumentation
  6. Product and Support Files
  7. Sample Preparation
  • According to my BioAnalayzer traces, my final library has a size distribution of 300 to 1500 bp. Will these large-sized fragments sequence? Should I perform further size selection on these libraries?
    • The Illumina Nextera Mate Pair Sample Preparation kit produces libraries with a broad size range distribution, typically between 300-1500 bp, with a peak centred around 600-700 bp. It is not necessary to size select the library further for sequencing purposes. Although the largest fragments may not cluster and sequence as efficiently, their presence in the library is not detrimental.
  • After what cycle will TruSeq control information be available?
      • Control information for a read is processed after alignment (cycle 25) but is not reported until cycle 52. For an indexed read, control information is reported after cycle 52 of R2.
      • If performing a run shorter than 52 cycles, you should see control reporting that is accurate for all controls except CTE, which will report counts in all band sizes.
  • Although there is an example result file and quantification with standard graph, I could not find the quantitative results of unknown samples. How do we see the quantitative results?
    • In the EcoStudy software click the Results tab. The well table on the right side of the window contains the quantification results. Quantity denotes the calculated quantity of each individual sample, and Average Quantity denotes the average quantity of all the replicates.

      You might need to resize or scroll the table to see all rows and columns. To resize the width, drag the vertical grey bar separating the table from the graph.

  • Are Ampure XP beads supplied with the TruSeq Sample Prep kits?
    • Ampure XP beads are user-supplied from Beckman-Coulter Genomics. See the appropriate TruSeq DNA or RNA sample preparation guide for ordering information.
  • Are FASTQ files generated in addition to BCL files?
    • BCL files are generated by RTA and FASTQ files are generated in post run analysis by MiSeq Reporter.
  • Are FFPE samples supported for TruSeq Custom Amplicon or the TruSeq Amplicon Cancer Panel?
    • We recommend FFPE-derived DNA only be used with TruSeq Custom Amplicon when using short amplicon lengths of 150 or 175 bp. Shorter amplicons provide better amplification than longer ones when the sample input is fragmented FFPE-derived DNA. For example, FFPE samples can be used with the TruSeq Amplicon Cancer Panel that has amplicons ~175 bp in length. Before starting a TruSeq Custom Amplicon prep with FFPE samples please be sure to use the Illumina FFPE QC Kit (WG-321-1001) to determine whether their samples meet the minimum quality criteria for inclusion in the assay. This protocol is qPCR based, where a Ct (threshold cycle) cut-off allows users to determine whether their samples will succeed in the TruSeq Amplicon assay or not. This measurement represents how well DNA amplifies and can be used to infer DNA quality. Testing has demonstrated that over 80% of samples passing the Illumina FFPE QC Kit protocol are expected to succeed in the TruSeq Amplicon assay.
  • Are FFPE samples supported for TruSight DNA Amplicon libraries?
    • No. FFPE DNA is not supported.
  • Are HiSeq/HiScanSQ flow cells compatible with the Cluster Station?
    • No. The HiSeq flow cell, also used on the HiScanSQ, must be clustered on a cBot equipped with an adapter plate suitable for the larger format of the flow cell.
  • Are MiSeq v3 kits compatible with all Illumina sample prep kits?
    • Yes. Just like v2 kits, MiSeq v3 kits are compatible with all Illumina sample prep kits. Some sample prep protocols require slight changes to reach optimum clusters. A concentration of 4 nM is recommended to begin the denature and dilute step before loading libraries for sequencing. Check the Illumina Tech Support Bulletin Board for the latest information on updates to sample prep protocols.

  • Are NextSeq 500 flow cells available in single-read and paired-end versions?
    • All NextSeq 500 kits include reagents for paired-end sequencing and a paired-end flow cell. However, single-read runs can be performed on a paired-end flow cell.

  • Are Nextera Enrichment kits supported on MiSeq?
    • Samples enriched with Nextera Enrichment kits can be sequenced on MiSeq. Full bioinformatics analyses may have to be performed offline, although a MiSeq Reporter workflow for enrichment samples is expected at a future date.
  • Are Nextera Rapid Capture Enrichment indexes and sequencing primers the same as the Nextera and Nextera XT sample prep?
    • Nextera Rapid Capture Enrichment index 1 (i7) and index 2 (i5) sequences are identical to index sequences used in other Nextera based kits. They use the Read 1 (HP10), Read 2 (HP11) and Index 1 (HP12) sequencing primers.

      Although index sequences are identical across Nextera kits, the index 2 primer reagents (i5, E501-E502) are not interchangeable across kits. Also, the Nextera Rapid Capture Enrichment kits use only two index 2 (i5, E501-E502) sequences compared to eight index 2 (i5) sequences used in other Nextera kits.

  • Are SYBR Green I Real-Time PCR assays less specific than TaqMan probe assays?
    • The specificity of any Real-Time PCR assay, whether TaqMan probe or SYBR Green I, is determined by the quality of the assay design. Non-specific amplification can occur for both SYBR Green I or TaqMan probe methods if the assay design is poor.

      TaqMan assays will not generate a signal for any non-specific amplification whereas SYBR Green I assays might. However, non-specific amplification will affect the amplification efficiency and sensitivity of TaqMan assays in the same way as SYBR Green I assays, even though the amplification is not detected.

      When designing primers for either system, it is important to avoid primer sets that generate any non-specific amplification products. With SYBR Green I assays, the ability to perform melt curve analysis is advantageous for primer design, as any non-specific amplification can be detected and identified in the melt curve. For TaqMan assays, detecting non-specific amplification usually requires another post-PCR analysis method such as agarose gel electrophoresis of the PCR products. Alternatively, the primers could be used in PCR with SYBR Green I and melt curve analysis performed after amplification to determine if any non-specific amplification occurs. This optimizes the primer design without the expense of a labeled probe.

  • Are TruSeq Custom Amplicon & TruSeq Amplicon Cancer Panel, Nextera XT applications supported on the HiSeq 2500/1500?
    • Sequencing runs of these library types are compatible with rapid run reagent kits on a HiSeq 2500 in rapid run mode. However, Illumina does not currently provide software for analyzing this type of data.
  • Are TruSeq Custom Amplicon, TruSeq Amplicon Cancer Panel, and Nextera XT applications supported with the HiSeq v4 mode?
    • Sequencing runs using these applications are compatible with v4 reagent kits and can be performed using the HiSeq v4 mode. However, Illumina does not currently provide software for analyzing this type of data.
  • Are TruSeq DNA adapters methylated?
    • TruSeq DNA v1 and TruSeq DNA, TruSeq DNA PCR-Free, and TruSeq Nano DNA LT sample prep kit adapters are methylated. However, TruSeq HT sample prep kit adapters are not methylated and are therefore not suitable for bisulfite sequencing applications.
  • Are TruSeq HT dual-indexed libraries single read or paired-end?
    • All TruSeq libraries are paired-end libraries.
  • Are TruSeq HT sample prep index sequences for the first 7 cycles of the first index read the same as TruSeq v2/LT kits, TruSeq Custom Amplicon kits, Illumina Nextera, etc.?
    • No, the TruSeq HT adapter plates contain 12 new and unique sequences that are used for the first index read. For specific index sequences, please refer to the Illumina Customer Sequence Letter , the Illumina Experiment Manager (IEM), or the appropriate sample preparation guide. The sequencing primer sequences are proprietary and not available.
  • Are TruSeq Targeted RNA Expression libraries compatible with MiSeq v3 reagents?
    • Yes, they are. Note that Targeted RNA Expression libraries require 50 cycles of sequencing (single read), plus dual indices.
  • Are UTR or promoter regions covered in the TruSight One Sequencing Panel?
    • TruSight One kits target the coding region only and do not include UTR or promoter regions. Currently only the Nextera Rapid Capture Expanded Exome covers UTR and promoter regions. 
  • Are clean‐ups/size selection steps necessary?
    • The protocol includes one Zymo cleanup step (following "tagmentation") and one Ampure XP size selection step (following limited cycle PCR). The final library should have a median insert size of ~250-300 to support long paired end 2x150 read lengths on the MiSeq system.

  • Are components from my other Nextera kits compatible with the Nextera Enrichment kit?
    • No. The Nextera Exome/Custom Enrichment kits contain unique reagents that are not provided in other Nextera kits, including Index 2 primers with an "E" prefix. Index 2 primers from other Nextera sample prep kits should not be used.
  • Are custom panels available for ordering?
    • Custom designed assays for Methylation profiling are now available to provide the ultimate flexibility of content! Please contact your Regional Account Manager to discuss your plans for custom designs.
  • Are data sets available for Nextera Rapid Capture Exome Enrichment?
  • Are degraded or FFPE samples compatible with the Nextera Rapid Capture Enrichment protocol?
    • The quality of DNA isolated from FFPE samples can be highly variable. Due to this variability, it is very difficult to reliably predict the quality of a library prepared from FFPE samples using the Nextera Rapid Capture Enrichment protocol. Illumina does not support FFPE or degraded DNA as input for Nextera Rapid Capture Enrichment. This does not mean that FFPE samples cannot be attempted, but that failed libraries originating from this sample type are not eligible for replacement or troubleshooting by Illumina.
  • Are degraded or FFPE samples compatible with the TruSight One protocol?
    • The quality of DNA isolated from FFPE samples can be highly variable. Due to this variability, it is difficult to predict the quality of a library prepared from FFPE samples using the TruSight One protocol. Illumina does not support FFPE or degraded DNA as input. FFPE samples can be attempted using the TruSight One protocol, but failed libraries originating from this sample type are not eligible for replacement or troubleshooting by Illumina.

  • Are designs equivalent between TruSeq Custom Enrichment, Nextera Custom Enrichment, and Nextera Rapid Capture Custom Enrichment projects?
    • Probe design algorithms have been optimized in DesignStudio for Nextera Rapid Capture Enrichment for better coverage across target regions, leading to fewer design gaps. Additional target probe spacing options have also been added for Nextera Rapid Capture Enrichment designed content.
  • Are flow cells provided in MiSeq v3 kits different from flow cells provided in MiSeq v2 kits?
    • No. The flow cells are identical. However, the upgrade to MCS v2.3 enables imagine of the flow cell in 19 tiles per surface. MCS v2.3 is required software for the MiSeq Reagent Kit v3.
  • Are libraries prepared with TruSeq Stranded RNA HT sample prep kits compatible with all Illumina sequencers?
    • Yes, these libraries can be used on all HiSeq, MiSeq, HiScanSQ and GA instruments.

  • Are my existing Illumina sequencing libraries compatible with the HiSeq?
    • Yes. Single read and paired end libraries for RNA and DNA applications are compatible with the HiSeq.
  • Are my saved projects from VariantStudio v2.1 compatible with VariantStudio v2.2?
    • Yes, VariantStudio v2.1projects are compatible with VariantStudio v2.2. Samples, annotations, including any imported custom annotations, and filter favorites are all carried over to VariantStudio v2.2.

  • Are other sequencing flow cells compatible with MiSeq?
    • No. The MiSeq uses a flow cell that is specifically designed for the MiSeq.

  • Are pre- and post- PCR areas required to run the Nextera Rapid Capture Enrichment protocol?
    • It is advisable to separate pre- and post-PCR due to amplicon generation in the protocol. For more information, see the Best Practices section of the Nextera Rapid Capture Enrichment Guide for additional information.
  • Are separate pre-PCR and post-PCR areas required for the TruSight DNA Amplicon assay?
    • Pre-PCR and post-PCR areas should ideally be in two separate rooms. If separate equipment is used (e.g., shaker, heat block, centrifuge) and a strict cleaning schedule is maintained, then TruSight DNA Amplicon can be performed in designated, non-adjacent areas in the same room. Regular bleaching is outlined in the User Guide. In addition, filtered pipette tips are also recommended.
  • Are separate pre-PCR and post-PCR areas required?
    • Pre-PCR and post-PCR areas should ideally be in two separate rooms. If separate equipment is used (e.g., shaker, heat block, centrifuge) and a strict cleaning schedule is maintained, then TruSeq Custom Amplicon can be performed in designated, non-adjacent areas in the same room. Regular bleaching is outlined in the User Guide. In addition, filtered pipette tips are also recommended.
  • Are somatic variants being called by the Isaac Variant Caller?
    • Only diploid variant calls are detected.
  • Are the HiSeq v4 upgrades compatible with HiScanSQ?
    • No. The HiScanSQ is not upgradable to use HiSeq v4 reagents.
  • Are the Illumina adapter sequences available?
    • Illumina adapter sequences are provided in the Illumina Customer Sequence Letter .
  • Are the RFID tags for the flow cell and reagent cartridge linked in any way?
    • No. The flow cell RFID is unique to the flow cell and reagent cartridge RFID is unique to the reagent cartridge.
  • Are the TruSeq Stranded RNA HT adapter plates compatible with the TruSeq RNA Sample Prep kit v1 or v2?
    • The adapter plate is not compatible with TruSeq RNA v1 or v2 kits as the protocol and the reagents are different.

  • Are the controls the same in the TruSeq DNA and RNA sample prep kits?
    • The same controls are used, but at different concentrations.

  • Are the data from GoldenGate Linkage V and/or Infinium HumanLinkage-12 projects compatible with linkage analysis software products on the market?
    • The output genotyping files from the GoldenGate and Infinium Linkage Panels are compatible with most linkage analysis software on the market. Details about Illumina’s software compatibility with downstream linkage analysis software can be found at Illumina’s website on the illumina.Connect program page.
  • Are the detector photomultiplier (PMTs) or photodiodes resistant to dark current, shock, and vibration?
    • Eco does not use PMTs or photodiodes, it uses a CCD camera as the detector which is designed to compensate for dark current.

  • Are the index sequences for the first 7 cycles of index 1 the same as current TruSeq Kits, TruSeq Custom Amplicon Kits, etc.?
    • No. For specific index sequences, please refer to the Illumina Experiment Manager (IEM) or the Illumina Nextera DNA Sample Preparation Guide.
  • Are the plates and seals for the Eco custom, or can I obtain consumables such as 8-tube strips or standard 48-well plates from other vendors?
    • The 48-well plates used in the system are custom designed for the Eco system, so you cannot substitute other plates or 8-tube (0.2 ml) strips. The plates are in a six-by-eight-well format with the same pitch and well size as standard 384-well Real-Time PCR plates. This means that you can use multichannel pipettes compatible with 384-well plates with the Eco plates.

      Eco plates and seals are only available through Illumina.

  • Are the recipes used on the HiSeq the same as on the Genome Analyzer?
    • No. Recipes used with HiSeq Control Software (HCS) are unique to HCS. Likewise, recipes used with the Genome Analyzer Sequencing Control Software (SCS) are unique to SCS.
  • Are there .bed files available for the Nextera Rapid Capture Exome and Expanded Exome Enrichment products?
    • Yes, the .bed files can be downloaded from the Nextera Rapid Capture Exome Enrichment Downloads support page.
  • Are there a minimum number of indices I need to use for TruSeq HT sample prep?
    • For dual indexing, at least 2 unique barcodes/indices need to be present for each Index Read. For less than 6 dual-indexed samples, Illumina recommends using single indexing.  Please refer to the pooling guidelines in the TruSeq sample preparation guide for the kit that you are using.
  • Are there any additional components I need to order to complete the prep?
    • In addition to either the Nextera XT DNA Sample Preparation Kit for 24 Samples (catalog# FC-131-1024) or 96 samples (catalog# FC-131-1096), there are corresponding Nextera XT Index Kits for 24 Indices/96 samples (catalog# FC-131-1001) and 96 Indices/384 samples (catalog# FC-131-1002). Please note that these indices are optimized for the Nextera XT kit and are not interchangeable with the Nextera kit.
  • Are there any analysis workflow changes for TruSeq Nano DNA libraries in comparison to TruSeq DNA libraries?
    • The TruSeq Nano DNA Sample Prep Kit uses the same workflow as TruSeq DNA Sample Prep Kit. No changes have been made in analysis.
  • Are there any differences in analysis for Nextera XT samples?
    • A new PCR Amplicon analysis workflow is available in MiSeq Reporter v1.3 (MSR) for the MiSeq system. The PCR Amplicon workflow requires specifying a manifest which is created in the Illumina Experiment Manager. A manifest is a list of all the targeted regions and their chromosome start and end positions. The manifest specifies regions of interest (ROIs) for the aligner and variant caller, which results in faster analysis times and visualization of results specific for only the ROIs. Note that CASAVA is not compatible with Manifest files. Please refer to the Illumina Experiment Manager User Guide (part # 15031335) for more information on sample sheet and manifest creation for Nextera XT libraries. The MiSeq software (MCS 1.2/MSR 1.3) can be downloaded from the Downloads tab of the Nextera XT Support page.
  • Are there any in-line controls in the sample prep?
    • No.
  • Are there any in-line sample prep controls in the TruSeq ChIP Sample Prep kit?
    • The TruSeq ChIP Sample Prep kit does not contain in-line controls due to the low input amount.

  • Are there any installation dependencies for VariantStudio?
    • VariantStudio requires the installation of .NET Framework 4.0 (Full, not 4.0 Client Profile). If .NET 4.0 is not detected, the installer requests that you install the framework. Another prerequisite for installation is the use of a 64-bit version of Windows.

  • Are there any performance differences (in terms of sequencing coverage) for GC rich amplicons?
    • Although most amplicons of interest will not likely be high GC-content, please note that coverage of high GC-content amplicons may be more variable as compared to other amplicons.
  • Are there any protocol changes when using v3 reagents and flow cell v3?
    • You need to increase your starting template concentration. However, the starting template volume (120 µl) is unchanged from previous kits.

  • Are there any protocol considerations for the TruSeq Stranded Total RNA Sample Prep Kits with Ribo-Zero Globin or Plant?
    • All Ribo-Zero kits follow the same protocol in the TruSeq Stranded Total RNA Sample Preparation Guide . The only component that differs in each kit variation is the rRNA Removal Mix.
  • Are there any protocol differences based on different amplicon lengths?
    • For amplicons greater than 500bp, Illumina recommends using a 0.6x AMPure XP cleanup (30 μl volume of beads). For amplicons less than 500bp, Illumina recommends using a 1.8x AMPure XP cleanup (90 μl volume of beads) to maximize yield.
  • Are there any safety considerations with the laser system on the HiSeq or HiScanSQ?
    • The HiSeq and HiScanSQ are Class 1 laser instruments as evaluated by IEC 60825-1 Edition 1.2. Under normal operation, the operator is not exposed to laser light.
  • Are there any special requirements for the master-mixed HiSeq v4 incorporation reagent?
    • Store the reagent at the recommended temperature labeled on the box and protect the reagent from natural and fluorescent light.
  • Are there any variations to the TruSight DNA Amplicon protocol when using panels of different amplicon plexities?
    • Yes. The number of PCR cycles has been optimized for the number of amplicons per reaction. The recommended number of PCR cycles for different amplicon numbers is indicated in the user guide and will ensure sufficient yield for the bead-based normalization while avoiding over-amplification.
  • Are there any variations to the protocol when using panels of different amplicon plexities and DNA inputs?
    • Yes. The number of PCR cycles has been optimized for various assay conditions taking into account both the number of amplicons per reaction as well as the type and amount of input DNA (e.g. FFPE). The recommended number of PCR cycles will ensure sufficient yield for the bead-based normalization while at the same time avoiding over-amplification.
  • Are there changes to reagents in the MiSeq v3 kits?
    • Yes. There are two new reagents in MiSeq v3 kits: new Incorporation Mix (IMT) and new Scan Mix (USM), and the PR2 volume is increased to 500 ml to support longer runs.

  • Are there considerations for combining certain TruSeq Small RNA indexes or only combining 1-2 indexes?
    • Testing has shown that performance shows minimal variability when combining low numbers of indexes, or across particular combinations of indexes, so any number or combination may be used.

  • Are there considerations for combining certain indexes or only combining 1-2 indexes?
    • Testing has shown that performance shows minimal variability when combining low numbers of indexes, or across particular combinations of indexes, so any number or combination may be used.

  • Are there different cBot kits for TruSeq HT dual-indexed libraries?
    • No, the cBot plates are the same; however, if sequencing TruSeq HT libraries on single-read flow cells, the TruSeq Dual Index Sequencing Primer Box, Single Read (single use box) (catalog# FC-121-1003) is necessary in order to sequence both index reads.

  • Are there duplicate samples in the Illumina iControlDB?
    • Samples are checked for duplicate barcode numbers, and duplicates are not allowed. This does not prevent the same sample from being run on different barcodes. There is not a check performed at the genotype level to ensure unique samples. Additionally, different sources may have run the same commercially available sources of DNA and submitted the data. Therefore, we recommend performing genotype-level comparison of the data that you are downloading prior to analysis to ensure the most accurate frequency information for your study.

  • Are there in-line controls included in the TruSeq Small RNA Sample Prep Kit?
    • There are no in-line controls for the TruSeq Small RNA Sample Prep Kit.

  • Are there in-line controls included in the small RNA kit?
    • There are no in-line controls for the small RNA kit.

  • Are there restrictions to the Maximum Number of Clusters the software can call?
    • There are no restrictions; however we recommend that the Maximum Number of clusters is set to match the biology of your samples.

  • Are there restrictions to the Minimum Number of Points in Cluster?
    • This number can be any number different from 0, however we recommend that the Minimum Number of Points in Cluster is set to match the biology of your samples and size of your dataset.  A general guideline is to set this number to 1-4% of the number of samples that are performing well in the data set.
  • Are there safe stopping points in the sample prep process?
    • Yes. You can safely stop this protocol after the ethanol precipitation or any Qiagen purification step. Store the samples at -15° to -20°C.
  • Are there third-party software programs that you can recommend for ChIP-Seq analysis?
    • There are a number of solutions available, including Bioconductor and MACS, which are available through Galaxy.
  • At what concentration are the DNAa Binding Dye primers supplied with NuPCR?
    • They are supplied in 4 µM.
  • BeadStudio allows for the customization of genotype reports (i.e. forward strand report, TOP strand report, design strand report, etc). What format is generated by iControlDB?
    • Genotypes generated by iControlDB are in the matrix format, and are forward strand reports.
  • Can CASAVA be used to analyze MiSeq data offline?
    • Yes, CASAVA can be used to analyze MiSeq data offline.  The MiSeq output folders are readable by CASAVA without a need to modify any configuration files.
  • Can DNA from FFPE samples be used?
    • Yes, the Cancer Panel has been optimized for use with DNA derived from FFPE sources. A simple qPCR-based quality control test (WG-321-1001, Illumina FFPE QC Kit) is recommended to ensure maximum sample success.
  • Can Eco be used to quantify proteins labeled with fluorescent dyes?
    • The Eco system does not support this application.
  • Can FFPE DNA samples be used for the GoldenGate Assay for Methylation?
    • While not specifically validated by Illumina Scientists, our commercial test sites observed good results with FFPE-derived DNA samples. Technical reproducibility is generally lower, with the r2 for Beta values ranging from 0.90 to 0.96.
  • Can GenomeStudio be used to view MiSeq data?
    • No. MiSeq Reporter and the Illumina Amplicon Viewer are used to view MiSeq data. For an overview of the software, see the MiSeq System software page.

  • Can GenomeStudio software display non-human sequence data?
    • Yes. GenomeStudio can display data from non-human genomes.
  • Can HiSeq X patterned flow cells be run on other platforms?
    • No. HiSeq X flow cells are designed and optimized specifically for use on the HiSeq X system. HiSeq X flow cells are not compatible with other HiSeq models.

  • Can HiSeq v4 and TruSeq v3 high output SBS reagents be combined together?
    • No. HiSeq v4 and TruSeq v3 high output SBS reagents cannot be combined. Each set of reagents has been formulated specifically for use only with its respective flow cell and instrument run parameters. The cluster kits and flow cells are also paired with respective kit types and cannot be used interchangeably.

  • Can I add custom genomic content to Illumina predesigned enrichment panels with Nextera Rapid Capture Custom enrichment?
    • Yes, the add-on workflow in DesignStudio for Nextera Rapid Capture Enrichment allows you to add 350–67,000 custom probes to Illumina pre-designed enrichment panels (approximately 80 Kb–15 Mb of custom genomic content). Get started with designing your project in DesignStudio .
  • Can I add genes or targets of interest to the Cancer Panel?
    • The Cancer Panel is a pre-synthesized, fixed content panel. As such, the content cannot be modified. However, the genomic targets from the cancer panel can be used as a starting point for a customized TruSeq Custom Amplicon design using DesignStudio.
  • Can I adjust the ramp rate to match various protocols?
    • Currently the ramp rate is not adjustable. Check back periodically and keep an eye on the monthly Illuminotes newsletter for updates.

  • Can I adjust the settings for cnvPartition?
    • Some settings for cnvPartition can be adjusted with a supplied configuration file. For information on adjusthing the configuration file, refer to the algorithm release notes. For more information on this algorithm, see the CNV Algorithms Technical Note.
  • Can I adjust what is in the Known Regions list?
    • Yes, this can be adjusted using Microsoft Excel. Information in any of the rows can be edited or deleted, or new rows can be added; however, no columns can be deleted. You can also create an entirely new known regions file and load it using the Filter Table interface. For more information about adjusting or creating new Known Regions lists, see the User Guide.

  • Can I analyze CIF files with BaseSpace?
    • It is not possible to analyze CIF files with HCS v2.2 on HiSeq v4 mode. The option to analyze CIF files is available in other modes.
  • Can I analyze rapid run data with CASAVA?
    • Yes, you can analyze rapid run data with CASAVA. If the zip BCL files option was chosen during run set up, you will need to use the bcl2fastq converter in place of the configureBclToFastq component of CASAVA. For rapid runs, you will align data from each flow cell separately and then merge the data at the configureBuild step.
  • Can I analyze v4 data with CASAVA?
    • Yes. You can analyze HiSeq v4 data with CASAVA. Note that you need to use the bcl2fastq v1.8.4 conversion software in place of the configureBclToFastq component of CASAVA.

      For HiSeq v4 runs, perform alignment of data from each separately, and then merge the data at the configureBuild step.
  • Can I assay all SNP classes ([A/T], [C/G], [A/C], [A/G], [T/C], [T/G]) with custom Infinium content?
    • Yes, all biallelic SNPs can be assayed using a combination of Infinium I and II probe designs.
  • Can I calculate PC and PPC errors in the PC Module?
    • No, this is not an option in the PC Module.

  • Can I change the design strand for individual targets or probes in Nextera Rapid Capture Custom Enrichment?
    • No. DesignStudio uses only the forward strand for designing Nextera Rapid Capture Custom Enrichment assays for optimal performance across the probe panel.
  • Can I combine HiSeq v4 SBS kits to achieve read lengths longer than what is provided in a kit?
    • Yes. See “Preparing SBS Reagents” in the HiSeq 2500 and HiSeq 1500 user guides for a list of how many SBS kits can be combined for supported run lengths.
  • Can I compare data between the TruSeq small RNA protocol and earlier Illumina small RNA protocols?
    • As with any molecular biology techniques, a change in protocol will give a change in absolute results. We do not recommend comparing absolute counts (or counts normalized to total counts) between different preparation protocols. However, comparison of fold change between the two protocols should yield good correlation.

  • Can I compare data from experiments with MCS3 to experiments with MCS4 plus RTE?
    • We have observed high concordance between detected probes using MCS3 versus MCS4 plus RTE and have found the variability in raw signal intensity correlations between MCS3 and MCS4 plus RTE to be comparable to the variability between freshly made MCS3 and MCS3 nearing the end of its shelf life. As is to be expected, signal intensity correlations and detected probe concordance decrease with input RNA quality.
  • Can I compare data from the TruSeq Stranded mRNA Sample Prep protocol to either the TruSeq RNA Sample Prep or the Directional mRNA-Seq Sample Prep unsupported protocol?
    • The data is comparable to TruSeq RNA Sample Prep v2 data. The TruSeq stranded RNA and Directional mRNA-Seq protocols are based on distinct ligation chemistries and the libraries they produce are expected to differ as a result. Direct sample to sample comparison is not recommended for these library types.

  • Can I compare expression data from different TruSeq Targeted RNA Expression runs?
    • Yes, if the data from different runs is normalized with same normalizer genes. The raw data from different runs cannot be merged together into a single dataset. For more information, see the MiSeq Reporter User Guide .
  • Can I create my own manifest file to analyze my Nextera Rapid Capture Enrichment data?
    • No. Nextera Rapid Capture Exome and Expanded Exome Enrichment manifests can be downloaded from the Nextera Rapid Capture Exome Enrichment Downloads support page.
  • Can I customize a HiSeq recipe?
    • With HCS v1.3 and later, you can customize a recipe to contain any number of reads. Reads can be indexed or non-indexed. However, Illumina does not guarantee the performance of custom recipes. Contact your Illumina Technical Support if you need assistance creating custom recipes.
  • Can I design for species other than human, mouse and rat with NuPCR?
    • DesignStudio allows user to submit sequences for design. Select Add New Target - Sequence. When submitting for species other than human, mouse, rat it is important to turn off "Specificity" checking against the source genome.
  • Can I design my custom Infinium BeadChip to detect chromosomal aberrations including Loss of Heterozygosity (LOH) and Copy Number (CN) changes?
    • Yes. However, it is important to design the chips with a sufficiently high density of SNPs to detect the changes of interest.
  • Can I design one single-plex reference gene assay and multiplex it with different target genes using NuPCR?
    • Yes, however this must be done my manually combining the two assays. Illumina recommends designing duplex assays using DesignStudio so the combination is screened bioinformatically.  
  • Can I designate a control lane using HiSeq Control Software (HCS)?
    • HCS allows you to designate a control lane during the run setup steps. Generally, you do not need to designate a control lane if the sequence you are analyzing has a balanced genome. In the case of an unbalanced or skewed base composition (e.g., bisulfite-treated samples) a control lane is recommended. This is not equivalent to a PhiX spike-in.
  • Can I do paired-end sequencing with mRNA-Seq libraries?
    • mRNA-Seq libraries are built with paired-end adapters and amplification primers. If you run them on paired-end flow cells, two paired runs of sequence data will be generated. However, these paired sequences can span unknown splicing junctions, which complicates alignment to a reference transcriptome. For this reason, Illumina does not support this application. However, users with bioinformatics experience can create their own alignment methods to handle these paired reads.
  • Can I download data from different sample types such as the HumanHap550 and the HumanHap300 in the same report?
    • Illumina strongly advises against this, as each product contains a different SNP list. Instead, download samples from a single array type and version number in each session.

  • Can I expect changes when using Sequence Analysis Viewer (SAV) with MCS v2.3 and MiSeq v3 reagents?
    • Intensity (Data by cycle) plots appear different due to non-linear exposure ramping. Non-linear ramping prevents exposure damage early in the read, which provides a boost later in the read when it is more necessary.

  • Can I expect comparable library preparation performance between the TruSeq DNA PCR-Free LT and HT kits?
    • Yes, both the LT and HT kits will give comparable results.
  • Can I expect comparable library preparation performance between the TruSeq LT and HT kits?
    • There are variances between the TruSeq LT and HT kits that may result in differences in yield and the amount of adapter dimer for the final library. Be sure to take extra care pipetting the AMPure XP beads accurately in the Clean Up PCR step, to minimize the amount of adapter dimer carried through to the final library. Also, note that the AMPure XP bead ratio at this step is optimized for the longer length of the dual-index adapters.

  • Can I expect comparable library preparation performance between the TruSeq Nano DNA LT and HT kits?
    • Yes, both the LT and HT kits will give comparable results; however, the LT kit is recommended for preparing less than 24 samples at a time.
  • Can I expect to see any changes in intensity with HCS v1.4 or HCS v1.5?
    • Intensity for the G channel is expected to be lower, but the rate of decay is much slower due to one of the new reagents in the TruSeq SBS Kit v3 - HS called SRE. Therefore, your data quality will not be impacted.

  • Can I export my Eco run data for use in other software packages?
    • You can export your data as multiple *.csv files or as a single Excel file with multiple worksheets. In addition, you can export a report as a PDF or PowerPoint file.

  • Can I freeze/thaw a TruSeq HT sample prep adapter plate?
    • Yes, the adapter plate can undergo freeze/thawing up to 4 times. More freeze thaws of the plates may impair assay performance.

  • Can I generate a call report?
  • Can I generate larger fragment sizes with TruSeq RNA sample prep protocols?
    • It is possible to use larger fragment sizes, however, we have found that shorter fragment sizes generate the best coverage. Please refer to Appendix A in the user guide for the TruSeq RNA sample prep kit that you are using for more information.

  • Can I get bead-level data?
    • Yes, bead-level data is available. Please contact Technical Support for assistance with this feature.
  • Can I get variant information (e.g., SNP, indel) from the TruSeq Targeted RNA Expression sequence data?
    • This information can be found in the .bam files. However, MiSeq Reporter will not perform variant calling or generate .vcf files. cSNPs can be analyzed and visualized using the Illumina Genome Viewer.
  • Can I have articles, publications, certificates of testing, or details on research studies using the Eco system that have been done on the instrument?
    • Since it is a new instrument that has not been released to market, there are currently no publications citing the use of the Eco system. Some data are available on www.ecoqpcr.com. Check back periodically and keep an eye on the monthly Illuminotes newsletter for updates.

  • Can I have both VariantStudio v2.1 and v2.2 installed on the same computer?
    • Yes. VariantStudio v2.2 and v2.2 can be installed concurrently on the same computer. The two versions of VariantStudio operate independently; information is saved in different application spaces. VariantStudio v2.1 installs as "Illumina VariantStudio 2.1", while v2.1 installs as "Illumina VariantStudio 2.2". Therefore, VariantStudio v2.2 does not overwrite the v2.1 installation. Using the default installation folder for both versions guarantees concurrent usability. If you decide to define a custom installation path, make sure that you do not use the same installation folder for both versions.

  • Can I hybridize Cy3-cDNA to my BeadChip?
    • The probe design and hybridization conditions were optimized for use with amplified cRNA. Varying the sample to cDNA would require re-optimization of every step of the protocol’s hybridization and wash conditions. Our protocol does not include a pre-block step of the microarray before adding sample. Adding Cy3-labeled sample would produce high background and poor results.

  • Can I import VCF files produced by non-Illumina software products into VariantStudio?
    • Yes. However, if analysis software other than Illumina analysis software is used to generate data, the VCF file might not contain the columns required by VariantStudio. See the VariantStudio User Guide (part # 15040890 B) for information about VCF file requirements.

  • Can I import a sample sheet?
    • Yes. Use a program like Excel to create a *.csv file. See the Eco Real-Time PCR System User Guide for instructions.
  • Can I import custom annotations?
    • Yes. You can import custom annotations to the software using the Custom Annotation function in VariantStudio. This feature requires that a custom annotation file uses a tab-delimited text format (TSV) and contains a header row that specifies the column names. VariantStudio allows both custom variant annotations and custom gene annotations.

  • Can I import data from non-Illumina platforms into GenomeStudio software?
    • No. GenomeStudio software is intended for use only with Illumina data.
  • Can I import raw intensity files (idats) into the PC Module?
    • No, it is not possible to generate a new project in the PC Module directly from idats. The polyploid workflow requires to first generate a genotyping project in the GenomeStudio Genotyping Module from the idats. The genotyping project (.bsc) can then be opened in the PC Module for polyploidy clustering.

  • Can I install Windows 7 updates on the MiSeq?
    • Updates to Windows are disabled. Illumina cannot guarantee system performance with future Windows 7 updates.
  • Can I load a project that contains multiple manifests?
    • No, KaryoStudio only accepts data from a single Infinium product at a time.
  • Can I modify the classification categories in VariantStudio?
    • Yes. By default, VariantStudio provides five classification categories: Pathogenic, Presumed Pathogenic, Unknown Significance, Likely Benign, and Benign. You can edit these categories through the Annotation & Classification | Classification Settings menu. 

  • Can I monitor my HiSeq run remotely?
    • Illumina provides the Sequencing Analysis Viewer (SAV) software that can be run on a Windows PC to remotely monitor your run. The software does not allow any control over the run and requires that the PC is connected to the analysis server over the network. Another application you can use to monitor your run is SeqMonitor , which allows you to monitor your run using your iPhone or iPad.
  • Can I obtain a SNP list?
    • Yes, the SNP list is available for download: 

      http://support.illumina.com/downloads/oncoarray-500k_product_files.ilmn.

  • Can I order a 300-cycle MiSeq v3 reagent kit separately from the TruSight One sample prep kit?
    • Three (3) MiSeq v3 reagent cartridges are included with the 9-sample TruSight One Sequencing Panel kit (FC-141-1006). If you need additional MiSeq reagent cartridges, it is necessary to order additional TruSight One 9-sample kits or MiSeq v3 600 cycle kits (MS-102-3003).
  • Can I order through MyIllumina FastTrack Services?
    • Yes, please use the following catalog numbers:

      FT-260-1002 for Infinium OncoArray FastTrack Service

      FT-260-1012 for Infinium OncoArray+ FastTrack Service (for the OncoArray plus add-on)                

  • Can I perform Single Read runs and still get both index sequences?
    • Yes. Please select the appropriate workflow based on the flow cell type (Single Read or Paired End) when starting your run in HCS.
  • Can I perform a primer rehyb on the MiSeq?
    • No, a primer rehyb is not possible on the MiSeq.
  • Can I perform comparisons across large groups of samples for cohort or population analysis?
    • No. Comparisons of two or more groups of samples cannot be performed using the application.

  • Can I perform family-based genetic disease analysis in VariantStudio?
    • VariantStudio supports family-based filtering of father, mother, affected child, and affected or unaffected siblings. In VariantStudio v2.2, the analysis requires input of the proband and one other sample, either a parent or a sibling. This process filters variants that are consistent with a particular inheritance mode, including autosomal dominant, autosomal recessive, X-linked recessive, and de novo mutation. 

  • Can I perform sample to sample comparisons?
    • Yes. Sample to sample comparison is done through the Cross Sample Subtraction Filter. This process filters variants that are present in one sample but not the other.

  • Can I perform single read runs and still get both TruSeq HT sample prep index sequences?
    • Yes. Please select the appropriate workflow based on the flow cell type (Single Read or Paired End) when starting your run so that the correct chemistry is used. If using a single-read flow cell the TruSeq Dual Index Sequencing Primer Box, Single Read (single use box) (catalog# FC-121-1003) is necessary in order to sequence both index reads.

  • Can I perform touchdown PCR with NuPCR?
    • Using touchdown conditions is not necessary as NuPCR exhibits excellent specificity.
  • Can I pool data from different TruSeq Targeted RNA Expression runs together if they used the same TOP pool?
    • No. Refer to the TruSeq Targeted RNA Expression tech note for information to determine the appropriate number of samples to be sequenced on a flow cell. For assays with low counts (< 10), pool fewer samples so that the read budget is increased for the low count samples.
  • Can I pool multiple libraries for a single Nextera Rapid Capture Enrichment reaction?
    • Up to 12 Nextera Rapid Capture Enrichment tagmented samples can be pooled and processed together for the enrichment part of the protocol. However, pooling is not performed with the Nextera Rapid Capture Exome Enrichment (8 rxn × 1 plex) Kit.
  • Can I process a sample with low sequence diversity?
    • RTA v1.18 includes optimizations to the algorithms that identify clusters and estimate the color normalization matrix and phasing and prephasing rates. These optimizations improve the ability of RTA to handle low-diversity samples, such as samples with unbalanced genome compositions (AT- or GC-rich genomes) or samples with low sequence diversity (amplicon sequencing). Because of these improvements, it is no longer necessary to designate a control lane in HCS to estimate matrix and phasing.

  • Can I quantify my cRNA by A260 instead of Ribogreen?
    • We have found quantification by RiboGreen to be more accurate than spectrophotometer readings. Contaminants from purification columns can give artificially high readings. If you must use A260, sample measurements should be > 0.5 to minimize the impact of contaminants on amplification.
  • Can I quantify my cRNA by A260 instead of Ribogreen®?
    • Illumina has found that quantification by RiboGreen is more accurate than spectrophotometer readings. Contaminants from purification columns can give artificially high readings. If you must use A260, sample measurements should be >0.5 to minimize the impact of contaminants on amplification.
  • Can I remove targets from Illumina predesigned Nextera Rapid Capture Enrichment panels?
    • The add-on workflow does not allow removal of targets from fixed content panels or previously ordered custom designed panels.
  • Can I reorder the same TruSeq Targeted RNA Expression oligo pool multiple times?
    • Yes. Your orders are saved in DesignStudio and can be reordered or used for an add-on project. If you need a large number of samples (> 10,000), contact Inside Sales.
  • Can I run FFPE samples on the Infinium Methylation Assay?
    • FFPE samples are NOT recommended for the standard protocol. FFPE samples are already highly degraded, with a high level of crosslinking, so conversion does not occur effectively. However, you can run FFPE samples on the Infinium HumanMethylation450 BeadChip using the FFPE automated or FFPE manual protocol along with the Infinium FFPE DNA Restoration Solution kit.
  • Can I run FFPE samples on the Infinium OncoArray 500K?
    • The FFPE restore kit has not been tested with the Infinium HTS assay and is not officially supported.  Customers attempting to run FFPE samples should be aware that they will have to recluster their FFPE samples together and may expect to see an increase in failed loci.
  • Can I run GenomeStudio software on a VMware virtual machine?
    • Yes. However, this is not officially supported by Illumina due to very slow performance.
  • Can I run Mate Pair libraries on the MiSeq?
    • Yes. However, the MiSeq Reporter software does not have an analysis workflow for Mate Pair libraries. To perform de novo assembly, set MiSeq Reporter to generate FASTQ files, and then export them into a de novo  assembler that will assemble Mate Pair libraries.
  • Can I run MiSeq Reporter on a computer independent of the MiSeq?
    • You can install a second licensed copy of the MiSeq Reporter on another computer. However, that computer must be connected to the same network as your MiSeq or the network location of the output folder. The computer must meet the following minimum hardware and software requirements: 64-bit PC with at least 8GB RAM (16-32 GB RAM for optimal performance), Windows Vista or Windows 7, and at least 1 TB of available hard disk space.

  • Can I run TruSeq HT libraries with Nextera libraries on the same lane or same flow cell? Are the indices the same or different?
    • Illumina does not support, and strongly advises against, running libraries prepared by different sample prep kits in the same lane of a flow cell. We do support running libraries prepared by different sample prep kits in different lanes of the same flow cell or spiking in llumina PhiX control library in the same lane as any user-prepared libraries. If different library types are run in different lanes, Dual Index Recipes and the Dual Index Primer Box must be used. The indices between TruSeq HT and Nextera are unique and not shared. Please see appropriate sample prep user guides for index sequences.

  • Can I run TruSeq Synthetic Long-Read DNA libraries on any Illumina sequencer?
    • Illumina currently supports sequencing of TruSeq Synthetic Long-Read DNA libraries on the HiSeq 1000, 2000, 1500, and 2500.
  • Can I run TruSight DNA Amplicon libraries on instruments other than the MiSeq?
    • Yes. TruSight DNA Amplicon libraries can be run on any Illumina sequencing platform but only the MiSeq provides a built-in analysis solution—MiSeq Reporter. NextSeq and HiSeq users can perform analysis using BaseSpace or BaseSpace Onsite, their own pipelines, or third-party tools. Amplicon workflows are expected mid-2014.
  • Can I run a control lane on a MiSeq flow cell?
    • No, a dedicated control lane is not possible because the MiSeq flow cell a single-lane flow cell. Illumina recommends using a spike-in method for adding a control to libraries.
  • Can I run both single read and paired-end recipes with TruSeq HT dual-indexed libraries?
    • Yes. However, for optimal coverage, Illumina recommends sequencing stranded RNA libraries with paired-end chemistry.

  • Can I run libraries prepared for the HiSeq or Genome Analyzer on the MiSeq?
    • Yes. You can run any library on the MiSeq provided your library has complementary adapters.

  • Can I run other applications while the Decode File Download Utility is running?
    • Yes. The application places practically no load on CPU or RAM, and has minimal impact on write speed to your hard disk.
  • Can I save CIF files in HiSeq v4 mode?
    • CIF files cannot be saved in HiSeq v4 mode. The option to save CIF files is available in other modes.
  • Can I save images from a run on the HiSeq or HiScanSQ?
    • No. Images are deleted automatically after they have been processed.
  • Can I save intensities from a run on the HiSeq or HiScanSQ?
    • Yes. The *.cif files can be saved and transferred to the analysis server over the network.
  • Can I save my filter settings as a template for use with other data sets?
    • Yes. After selecting filter settings using options in the Filters pane, you can save the filter for later use. To apply a saved filter, click the drop-down menu in the Current field, and select a filter name from the list. The filter is automatically applied.

  • Can I sequence Nextera Rapid Capture Enrichment libraries on the Genome Analyzer IIx?
    • Yes. When preparing libraries on the cBot and sequencing on the HiSeq 2000, GAIIx, and in High Output mode on the HiSeq 2500 new primers are required whether performing a non-indexed, single-indexed, or dual-indexed run. Use the TruSeq Dual Index Sequencing Primer Kit for Paired End runs (catalog # PE-121-1003), which is good for a single run and contains the required primers for sequencing (HP10, HP11, HP12). These primers are included with MiSeq and HiSeq 2500 rapid run reagent kits.
  • Can I sequence Nextera Rapid Capture Enrichment libraries on the same flow cell with other Nextera libraries?
    • Yes. All Nextera based libraries use the same sequencing and index primers (HP10, HP11, HP12) and can be loaded in different lanes of the same flow cell for sequencing.
  • Can I sequence TruSeq Custom Enrichment libraries on the MiSeq?
    • Yes, you can sequence your enriched libraries on the MiSeq System, obtaining results in hours instead of days. However, the MiSeq Reporter currently cannot process the manifest file provided for your enrichment experiment. Therefore, the MiSeq Reporter maps your reads to the entire genome, not just the enriched regions, and alignment of the reads takes at least four hours.

      Prepare your libraries as described in the protocol for TruSeq Exome Enrichment Kit or the TruSeq Custom Enrichment Kit, and prepare your libraries for sequencing as described in the MiSeq System User Guide. When you are ready to set up your sample sheet using the Illumina Experiment Manager, select the following settings:

      • Select Workflow: select MiSeq Reporter and the Resequencing workflow.
      • Select Compatible Assay: select TruSeq DNA/RNA.

      MiSeq Reporter will generate the standard Resequencing reports. Consequently, you will need to zoom in on your enriched regions to obtain relevant data.

  • Can I sort the data displayed in iControlDB?
    • Yes, you can simply click the header of any of the columns and it will sort that column for you.
  • Can I start with mRNA for TruSeq Targeted RNA Expression?
    • Use 0.5–2.5 ng of optimized mRNA input. However, starting with mRNA input has not been tested and is not currently supported.
  • Can I still order individual components?
    • Current kit configurations include all components, and are orderable via a single catalog number. See this web page for more information: http://www.illumina.com/pagesnrn.ilmn?ID=70
  • Can I study copy-neutral LOH with KaryoStudio? Can cnvPartition do this?
    • Yes, it is possible to study copy-neutral LOH with KaryoStudio and cnvPartition. To ensure that you have the latest version of the cnvPartition algorithm, contact Illumina Technical Support (technicalsupport@illumina.com ). Note that the amount of copy-neutral LOH present across a typical genome can be quite large. Illumina recommends setting the filter to a large size in order to limit the number of regions found by the algorithm.
  • Can I submit custom designs for species other than human or mouse?
    • Illumina does not currently have custom design support for this product.
  • Can I switch modes on only one side of my HiSeq system?
    • Perform mode switching procedures on the side that you intend to perform the sequencing run in the new mode. For example, if you have just completed two rapid runs on side A and B, and would like to set up only one high output flow cell on side A, change the mode for only side A.

      A high output run on side B cannot be performed until a mode change is complete on side B. For efficiency and the most run flexibility, perform mode switching on both sides of the system at the same time.
  • Can I thaw the reagent cartridge in the refrigerator overnight?
    • For best results, thaw the reagent cartridge in a room temperature water bath for approximately 60 minutes.
  • Can I transfer called SNPs from CASAVA to use in custom BeadChip design?
    • Called SNPs from CASAVA can be parsed to ADT via two reports: a dbSNP Report for the start and stop positions of the region flanking the SNP, and a Sequence Report which provides the actual sequence of the region flanking the SNP.
  • Can I upload copy number variation data?
    • No. Only genotype information is contained within the iControlDB at this time.
  • Can I upload data from my custom GoldenGate OPA or my custom iSelect product?
    • The iControlDB is intended only for standard content Illumina products. BeadStudio will allow you to upload your data from a custom project, but this data will not pass the validation stage and will not be released to the database.
  • Can I use BeadStudio to download data from the database?
    • No. Only the Illumina iControlDB - Client program can be used to download data from the database.
  • Can I use DNA extracted from saliva?
    • While we do not have experience using DNA extracted from saliva using the GoldenGate Assay, internal testing was performed using the Infinium I Assay with DNA extracted from saliva with a kit from DNA Genotek. The call rates and reproducibility from this test were > 99%. We have not tested kits for DNA extraction from saliva from any other source.
  • Can I use FFPE-derived total-RNA?
    • We performed this protocol on FFPE-derived total RNA with good results. The poly-A purification step efficiently removes the ribosomal RNA in the sample, and the highly degraded transcripts that are present are purified by binding to the Poly-T beads. The cDNA fragments created from FFPE will be only as long as the length of mRNA that is still attached to the poly-A tail in each molecule. For example, if the RNA purified from an FFPE sample is degraded to an average length of 200 bps, then you will only get reads in the last 200 nts of most mRNA molecules in that sample.
  • Can I use Formalin-Fixed Paraffin-Embedded (FFPE) samples in the GoldenGate Assay?
    • The GoldenGate Assay can tolerate relatively short stretches of target DNA (> 200 bp) and can be quite tolerant of degraded FFPE samples. Internal experience with FFPE samples used with the GoldenGate Assay indicates that high-quality genotyping data can be obtained. Decreased call rates from FFPE samples compared to genomic DNA samples may be observed, but the decrease in call rate depends on the level of sample degradation.

  • Can I use GenomeStudio to download data from the iControlDB database?
    • No. Only the Illumina iControlDB-Client program can be used to download data from the database.
  • Can I use HiSeq or Genome Analyzer reagents on the MiSeq?
    • No. MiSeq kits use an all-inclusive reagent cartridge system that is specifically designed to deliver the reagents necessary for cluster generation, sequencing, and paired-end chemistry.

  • Can I use Illumina VariantStudio with data from other sequencing platforms?
    • No. The Illumina VariantStudio End User License Agreement (EULA) states that Illumina VariantStudio must be used solely to analyze data generated from an Illumina sequencing instrument.

  • Can I use LCM-derived total-RNA?
    • While this might be possible with as little as 100 ng with high-quality total RNA, Illumina does not have direct experience with this method.
  • Can I use MiSeq Reporter software to analyze TruSeq Custom Amplicon and TruSeq Amplicon Cancer Panel data from a HiSeq system?
    • No. File directory structures from a HiSeq system are incompatible with MiSeq Reporter software.
  • Can I use MiSeq Reporter software to analyze TruSight DNA Amplicon data from a NextSeq or HiSeq system?
    • No, file directory structures are incompatible with MiSeq Reporter software.
  • Can I use Nextera DNA or Nextera XT DNA sample prep kits with the Nextera Rapid Capture Enrichment reagents?
    • No. The Nextera Rapid Capture Enrichment reagents and protocol have been optimized to only be used together. The kits include all components for both library prep and Rapid Capture.
  • Can I use NuPCR even if I do not own an Eco PCR machine?
    • Yes, NuPCR works on all instruments from major manufacturers. It has been shown to work on the following instruments:

      • Eco Real-Time PCR System
      • Applied Biosystems 7900HT
      • Applied Biosystems 7500
      • Applied Biosystems ViiA 7
      • Bio-Rad CFX 384
      • Qiagen Rotor-Gene
  • Can I use Sequencing Analysis Viewer (SAV) to view data from a HiSeq X run?
    • Yes. SAV 1.8.37 is required to view data from a HiSeq X run. You can download SAV for use on a networked computer or use SAV in BaseSpace. SAV 1.8.37 is also compatible with other HiSeq models, NextSeq, and MiSeq run data.

  • Can I use Sequencing Analysis Viewer (SAV) to view data from a NextSeq run?
    • Yes. SAV 1.8.36 is required to view data from a NextSeq run. You can download SAV for use on a networked computer or use SAV in BaseSpace. SAV 1.8.36 is also compatible with HiSeq and MiSeq run data.

  • Can I use SuperScript III instead of SuperScript II?
    • Illumina recommends following the protocol as closely as possible. The protocol was optimized with SuperScript II.
  • Can I use Zymo beads instead of sample purification beads for Nextera Rapid Capture Enrichment clean up steps?
    • Nextera Rapid Capture Enrichment kits include a sufficient volume of Sample Purification Beads regaent to process the intended number of samples for the kit. Illumina does not recommend the use of beads from Zymo or any other manufacturer with this protocol.
  • Can I use a Covaris E210 or E220 instead of the S2 or S220 sonicators?
    • The Nextera Mate Pair protocol has been validated with the S series Covaris sonicators. According to Covaris, the performance of the E series (E210 and E220) should be similar when using the equivalent settings (if using the E210, follow the settings recommended for the S2; if using the E220, follow the settings for the S220). However, please note that these settings may not be directly transferable and using a Covaris outside of the S-series as recommended in the protocol will likely require optimization.
  • Can I use a TruSeq DNA sample prep kit with a TruSeq DNA PCR-Free sample prep protocol? Can I use a TruSeq DNA PCR-Free sample prep kit with a TruSeq DNA sample prep protocol?
    • No, kits are not interchangeable.
  • Can I use a TruSeq HT sample prep kit with a TruSeq LS sample prep protocol? Can I use a TruSeq LT sample prep kit with a TruSeq HS sample prep protocol?
    • Yes, the low sample (LS) and high sample (HS) workflows may be applied with both the high throughput (HT) and low throughput (LT) kit configurations. Please refer to the user guide for the TruSeq sample prep kit that you are using for Illumina's recommendations on kit and protocol combinations to ensure the best results.

  • Can I use a home-made agarose gel instead of an E-Gel for TruSeq Synthetic Long-Read DNA library prep?
    • The protocol was optimized with E-Gels. Typical agarose gel dyes (stain) interfere with size selection and downstream PCR.
  • Can I use a partial plate for an initial run, and then resuse the empty wells from that same plate for a second run?
    • Illumina does not recommend reusing plates as there is a significant risk of contamination.

  • Can I use a thermal cycler in place of a microheating system for tagmentation in the Nextera Rapid Capture Enrichment protocol?
    • Incubation temperature and times have been established based on using the required MIDI plate and TruTemp microheating system. However, efficient tagmentation can take place using a thermal cycler and compatible 96-well plate. However, because of the additional sample transfers and requirement for manual pipette mixing, some degree of sample loss can incur which can negatively impact library diversity.
  • Can I use a thermal cycler in place of a microheating system in the TruSeq Targeted RNA Expression protocol?
    • Yes. However, the samples must be transferred to a MIDI plate for the Wash, Extend, and Ligate Bound Oligos procedure, then transferred to a PCR (HSP) plate for the PCR Amplification procedure.
  • Can I use cRNA prepared according to Affymetrix's protocol on BeadChips?
    • Illumina has had poor results when using fragmented sample on our arrays. The Affymetrix protocol requires fragmentation during sample preparation due to the short probes used on their arrays as it reduces the secondary structure of RNA. Illumina's longer probes allow for more stringent hybridization conditions which preclude the need for RNA fragmentation. If you have labeled sample which has NOT been fragmented, you may obtain satisfactory results, depending on the age and quality of the sample.
  • Can I use degraded total RNA for mRNA-Seq library production?
    • While it is possible to create libraries for sequencing on the Genome Analyzer from degraded RNA, it will have a large effect on the performance of the assay. One of the first steps in the process is the purification of poly-A mRNA using a poly-T capture step. If the RNA is degraded, the mRNA that is captured at this step will not be full length, and will not give full-length cDNA products after random priming. High-quality total RNA produces even, end-to-end coverage of each mRNA molecule. If the RNA is degraded, you might observe a noticeable 3'- to 5'-bias in the number of reads for most transcripts. Greater degradation will result in greater bias.

      When you compare expression levels across many samples, compare counts only from samples that have a similar RIN (RNA integrity number) from the Agilent Bioanalyzer to give the most quantitative results.

  • Can I use different sequencing primers in the two Rapid flow cell lanes?
    • No. The same sequencing primer is distributed across both lanes of a Rapid flow cell as part of the HiSeq 2500 onboard cluster generation workflow.
  • Can I use input RNA from FFPE tissues for TruSeq Targeted RNA Expression?
    • Yes, Illumina recommends using 100-200 ng degraded or FFPE samples with an average fragment size of ≥ 200 bp for optimal hybridization. In addition, check the total RNA integrity following isolation on an Agilent Technologies 2100 Bioanalyzer using an RNA 6000 Nano Chip. For more information, see the RNA Input Recommendations section of the TruSeq Targeted RNA Expression Guide .
  • Can I use only one of the indices of a dual-indexed library?
    • Sample prep for dual-indexed libraries requires that both indices be present on the library. However, the second index does not need to be read during sequencing. A single-indexing workflow is supported on Illumina sequencing instruments, where only Index 1 is used. See the instrument user guide for more information about setting up an eight-base single-indexed sequencing run.

  • Can I use taq polymerase from other vendors with NuPCR Assay?
    • No, NuPCR is optimized for the enzyme in the NuPCR Master Mix.  
  • Can I use the GoldenGate Linkage panel with samples from many ethnic groups?
    • Yes. We provide a list of the markers in the Linkage V Panel and include minor allele frequency information for samples from the three major ethnic groups represented by the HapMap samples. The panels were constructed to optimize information content across Caucasian samples, and it has been augmented to increase information content for the African population.
  • Can I use the HiSeq X reagents on my other HiSeq system?
    • No. HiSeq X reagents are specifically designed and optimized for HiSeq X systems. HiSeq X reagents are not compatible with other HiSeq models.

  • Can I use the Nextera XT kit on all Illumina sequencing platforms?
    • Although the Nextera XT kit has been optimized for use on the MiSeq system, this kit can be used on any Illumina sequencing platform. However, please note that CASAVA is not compatible with Manifest files and therefore is unable to analyze data generated on the HiSeq, HiScanSQ or GAIIx platforms.
  • Can I use the TruSeq DNA HT Sample Prep Kit to generate libraries from amplicons and FFPE samples or only from genomic DNA?
    • All TruSeq DNA sample prep kits, including the HT version, supports input of standard genomic DNA only. Illumina offers other kits for Amplicon library prep. The TruSeq DNA HT Sample Prep kit supports high quality genomic DNA as input for sample prep to ensure best results. Please see the DNA Input Recommendations section of the TruSeq DNA Sample Preparation Guide for further information.

  • Can I use the TruSeq HT adapter plate to generate just a few samples at a time?
    • The adapter plate is single-use per well and Illumina recommends that it not undergo more than four freeze/thaw cycles. For this reason, Illumina strongly recommends processing at least 24 samples at a time with the TruSeq HT sample prep kit and adapter plate. Even if 24 samples are processed at a time, these 24 samples can be pooled and sequenced in any Illumina supported configuration including low-plex. If it is desired to prepare only a few samples at a time, Illumina recommends purchasing a TruSeq LT sample prep kit.

  • Can I use the TruSeq Stranded RNA HT sample prep kits to generate libraries from FFPE samples or from only intact total RNA?
    • The TruSeq Stranded mRNA HT Sample Prep Kit supports high quality RNA as input for sample prep as the protocol relies on a poly-A tail purification step. The TruSeq Stranded Total RNA HT Sample Prep Kit is compatible with FFPE samples. Please see the RNA Input Recommendations section of each kit's user guide for more information.
  • Can I use the Zymo EZ DNA Gold kit for bisulfite conversion?
    • Yes, you can, but you must use this kit for your entire project. That means you cannot use a mixture of samples that have been converted with different kits on the same arrays, or within the same project.

  • Can I use whole-genome amplified (WGA) DNA for GoldenGate genotyping?
    • Please refer to our technical note entitled Using Whole-Genome Amplified (WGA) DNA Samples in the GoldenGate Genotyping Assay (PDF) before conducting your genotyping experiment. You can also find a link to the publication referenced in the technical note here: Two Methods of Whole-Genome Amplification Enable Accurate Genotyping Across a 2320-SNP Linkage Panel.
  • Can I use whole-genome amplified (WGA) samples with the Infinium Assay?
    • Illumina does not recommend using WGA samples as input for the Infinium Assay as the first step in the protocol is a WGA step. Up to a 4% decrease in call rate was observed in the limited internal testing performed. The decrease in call rate will vary depending on the specific sample and WGA method used. Additionally, any allelic bias present in the original WGA sample may be compounded by another WGA reaction.
  • Can I use whole-genome amplified (WGA) samples with the Infinium HD Assay?
    • Illumina does not recommend using WGA samples as input for the Infinium HD Assay because the first step in the protocol is a WGA step. Our scientists observed up to a 4% decrease in call rate during limited internal testing. The decrease in call rate varied depending on the specific sample and WGA method used. Additionally, any allelic bias present in the original WGA sample might be compounded by another WGA reaction.
  • Can I view data from more than one sample at a time in the ICB?
    • Yes, data from a second or third sample can be plotted concurrently by selecting Settings | Trio View from the menu.
  • Can Illumina Nextera DNA Sample Preparation kits be used for methylation studies?
    • No, because the adaptors are not methylated.
  • Can LOH (Loss of Heterozygosity) / CN (Copy Number) analysis be performed with GoldenGate genotyping?
    • LOH and CN changes have only been validated using the Infinium Assay. We do not support interpretation of LOH/CN data on the GoldenGate Genotyping Assay. Custom assay pools (OPAs) must provide high density and, therefore, high resolution to generate meaningful data for LOH and CN analysis.
  • Can Nextera DNA Sample Preparation kits be used in TruSeq Exome Enrichment/Custom Enrichment?
    • This is not supported at this time.
  • Can Nextera Rapid Capture Enrichment libraries be run on the MiSeq System?
    • The MiSeq provides sufficient output to run a single Nextera Rapid Capture Enrichment per MiSeq run. However, the HiSeq 2500 continues to be the most cost effective and highest throughput platform for exome sequencing.

  • Can Nextera dual-indexed libraries be combined with other library types on the same flow cell?
    • Yes. If you are sequencing combined single-indexed and dual-indexed libraries on the HiSeq or GA, use sequencing primers provided in the TruSeq Dual Index Sequencing Primer Box (Single Read, Catalog # FC-121-1003 or Paired End, Catalog # PE-121-1003). Primers provided in this kit are compatible with all library types. Single-indexed libraries and dual-indexed libraries need to be loaded onto separate lanes of the flow cell. For more information, see Sequencing Mixed Libraries on a HiSeq or GA Flow Cell .
  • Can Nextera handle sample indexing, and how many indices does Nextera support?
    • If you would like to index Nextera prepared samples, there are two Nextera Index Kits: a 24 Index Kit with sufficient reagents for 96 samples (Catalog # FC-121-1011) and a 96 Index Kit with sufficient reagents for 384 samples (Catalog # FC-121-1012).
  • Can RiboZero be used before sample prep and enrichment in the TruSeq RNA Access Library Prep protocol?
    • The use of RiboZero before sample prep and enrichment is not necessary and has not been tested by Illumina. The initial RNA input is very low for total RNA (10 ng) and FFPE RNA (20 ng).
  • Can Sequencing Analysis Viewer (SAV) be used with the MiSeq to view primary analysis results?
    • Yes. You can install SAV on another computer that is connected to the same network as the instrument. MiSeq data will appear when you select All or Lane 1 from the drop-down menu. For more information, see the Sequencing Analysis Viewer User Guide .
  • Can TruSeq DNA HT (dual-indexed) libraries be used for downstream enrichment using the TruSeq Exome Enrichment or TruSeq Custom Enrichment kits? Are there any differences if the HT dual-indexed libraries are used compared to the TruSeq DNA v2/LT kits?
    • Yes, TruSeq DNA HT libraries can be used for downstream enrichment using either the TruSeq Exome Enrichment or TruSeq Custom Enrichment kits. There are slight differences in the design of the v2/LT and HT adapters; therefore, use of the HT adapters may result in slightly lower percent enrichment compared to the v2/LT adapters.

  • Can TruSeq DNA PCR-Free libraries be used for TruSeq Enrichment?
    • No, the yield from TruSeq DNA PCR-Free libraries is not sufficient as input into TruSeq Exome or Custom Enrichment assays.
  • Can TruSeq Exome Enrichment Kits be used for methylation applications?
    • The TruSeq Exome Enrichment kit protocol is not currently compatible with bisulfite sequencing.
  • Can TruSeq HT prepared libraries be combined with other library types, such as TruSeq v2/LT, in the same lane?
    • Illumina does not support running libraries prepared by different sample prep kits in the same lane of a flow cell and does recommend running any combination of library types in a single lane. Please note that Illumina PhiX can be spiked in to any lane with user-prepared libraries.

  • Can TruSeq Synthetic Long-Read DNA libraries be combined with other library types on the same flow cell?
    • Yes, a sample sheet is used to specify how each lane is to be analyzed. However, it is critical that TruSeq Synthetic Long-Read DNA library sequencing parameters (2 x 101 + 8 bp index) apply to the entire flow cell. If multiple TruSeq Synthetic Long-Read DNA libraries are run on the same flow cell, each must be run on a separate lane, because the same barcoding scheme is utilized for each sample.
  • Can TruSeq Synthetic Long-Read DNA size-selected samples be stored frozen?
    • No, they can be stored at 2°C to 8°C for up to 3 months. Freezing long fragmented DNA can lead to significant strand breakage leading to poor results, such as low or no yield, and short N50.
  • Can TruSeq adapters be used for methyl-Seq?
    • TruSeq adapters are methylated.  However, the kits are not currently compatible for the following reasons:

      • WGBS:  The PMM (polymerase mix) used in the TruSeq DNA Sample Prep kits cannot handle uracil bases in its template.  This means that bisulfite-converted DNA cannot be processed directly with TruSeq kits, unless another polymerase is used to amplify the samples.
      • RRBS: Same as above, and in addition the length of TruSeq adaptors results in the generation of adaptor dimers that overlap the size range of the targeted DNA, making them incompatible with this application.        
  • Can TruSeq v3 reagents be used on a system that has been upgraded for HiSeq v4?
    • Yes. You can continue to use TruSeq v3 reagents as well as HiSeq v4 reagents. The chemistry type is specified when you select the mode before starting the run.
  • Can TruSight One libraries be run on the MiSeq System?
    • Yes, the TruSight One Sequencing Panel is designed to allow trio sequencing and data analysis using the MiSeq system.  The 9-sample TruSight One kit is intended for use with the MiSeq and contains three MiSeq v3 reagent cartridges.  The 36-sample kit does not contain sequencing reagents and can be sequenced on any Illumina sequencer.

  • Can VariantStudio be used in a clinical lab, such as a CLIA lab?
    • Yes, but only after validating the software according to Institution, Local, State, and Federal guidelines before using the Illumina VariantStudio software.
  • Can a USB device be plugged into the MiSeq during the run?
    • Illumina recommends that you wait until the completion of a run before inserting a USB device into the MiSeq.
  • Can a run be paused while the MiSeq is sequencing?
    • Yes, a run can be paused briefly. The MiSeq Control Software (MCS) will pause the run and place the flow cell in a safe state. When you resume the run, the software resumes from the point that it was paused.
  • Can all 96 indexes support use of demultiplexing with mismatch=1?
    • Yes, the nucleotide distance of all the indices is such that mismatch of 1 still makes a unique index.

  • Can custom primers be used on the MiSeq?
    • Yes. The MiSeq reagent cartridge includes three empty reservoirs for custom primers. You have the option of using a custom primer for Read 1, the Index 1 Read, and Read 2. For more information, see Using Custom Primers on the MiSeq (part # 15041638).

  • Can custom primers be used on the NextSeq 500?
    • Yes. There are three reservoirs on the NextSeq reagent cartridge reserved for custom primers: #7 for a custom Read 1 primer, #8 for a custom Read 2 primer, and #9 for a custom Index 1 primer.

  • Can data from Illumina Nextera libraries be directly compared to legacy Nextera kits?
    • The Illumina Nextera kit has been optimized and performances between kits may be different. For example, the new Illumina Nextera DNA Sample Preparation kits exhibit improved GC performance compared to the legacy kits.
  • Can dual-indexed libraries be combined with other library types in the same flow cell?
    • Illumina supports sequencing libraries prepared by different sample prep kits such as single-indexed libraries and dual-indexed libraries in different lanes of the same flow cell. To do this, you must use a dual-indexed workflow and the appropriate dual-indexed primers, and prepare two sample sheets. For more information, see Sequencing Mixed Libraries on a HiSeq or GA Flow Cell.

  • Can genotyping projects that were opened and modified in the PC Module be imported back into the GenomeStudio Genotyping Module?
    • No, a polyploid project (.pcm) can only be opened using the PC Module.

  • Can index reads on TruSeq dual-indexed libraries be rehybridized?
    • For paired-end flow cells, only Index 1 can be re-hybed, and this must occur before Index 2 begins. For single-read flow cells, Index 1 and Index 2 can be rehybridized before the run completes.

  • Can larger DNA inserts be selected during the gel size selection step of the TruSeq ChIP Sample Prep protocol?
    • The protocol is optimized for the inclusion of a gel size selection step selecting for a DNA insert of 150–200 bp. Longer inserts could be used, however, this may decrease the signal to noise for the binding motif and may require modification for optimal run performance.  Illumina recommends selecting a gel size range of 250–300 bp for the final library.

  • Can low numbers of indices/pooled samples be run together? What will happen if I run improper index combinations? How do I choose the most appropriate combination of indices?
    • If pooling fewer than the number of indices provided in the kit, it is necessary to consider low-plexity index combinations. Color balance during the index read is needed to ensure proper image registration. If there is no signal in one of the color channels (red or green) of the index read, image registration may fail and no base will be called for that cycle. If no base is called, the index read may not be able to be matched to the sequence specified in the sample sheet, and then samples will not be able to be demultiplexed. Please see the Pooling Guidelines section in your TruSeq sample preparation guide for instructions. The Illumina Experiment Manager (IEM) will give warnings when generating HiSeq, HiScanSQ, or GA sample sheets if the index combinations do not meet this requirement.

  • Can my cBot cluster both HiSeq and Genome Analyzer flow cells?
    • Yes. The cBot can cluster both HiSeq and Genome Analyzer flow cells. However, you must use the appropriate flow cell adapter plate to accommodate the different dimensions of the flow cells. Dedicating a cBot to one platform avoids routinely switching adapter plates.
  • Can my sample report templates be used in other VariantStudio projects?
    • Yes. Report templates created and saved in VariantStudio are available to other VariantStudio projects.

  • Can other thermal cyclers be used with the TruSeq Nano DNA Sample Prep protocol?
    • Coverage of GC regions can be impacted by the model, settings, and performance of the thermal cycler used. Illumina has validated the Bio-Rad DNA Engine Tetrad 2, the Bio-Rad S1000, and the MJ Research PTC-225 DNA Engine Tetrad thermal cyclers. Other thermal cyclers may differ in their performance across the genome.
  • Can qPCR be used to quantitate small RNA libraries?
    • Yes. For the most accurate measurements, the control library used to generate a standard curve for qPCR should be as similar as possible to the library being measured, which makes it necessary to use a small RNA library as a control library.
  • Can rapid run SBS reagents and high output SBS reagents be combined together?
    • No. Rapid run SBS reagents and high output SBS reagents cannot be combined. Each set of reagents has been formulated specifically for use only with its respective flow cell and instrument run parameters. The cluster kits and flow cells are also paired with respective kit types and cannot be combined.
  • Can samples be stored after DNA activation?
    • Once precipitated and resuspended as per the user guide, DNA can be stored at 4°C overnight or -20°C for up to six months.
  • Can samples generated with the TruSeq Stranded Total RNA Sample Prep Kits with Ribo-Zero Globin or Plant be multiplexed in the same lane as human TruSeq RNA samples?
    • Yes, but the indices are the same. Make sure that all samples have different index combinations.
  • Can samples processed with the MCS3 reagent and the MCS4 plus RTE reagent be analyzed within the same GenomeStudio project?
    • Yes, samples can be analyzed in a single experiment. We have observed high concordance between detected probes using MCS3 versus MCS4 plus RTE and have found the variability in raw signal intensity correlations between MCS3 and MCS4 plus RTE to be comparable to the variability between freshly made MCS3 and MCS3 nearing the end of its shelf life.  As is to be expected, signal intensity correlations and detected probe concordance decrease with input RNA quality.  

  • Can secondary analysis by MiSeq Reporter be delayed after a sequencing run completes?
    • No, secondary analysis cannot be delayed automatically. Secondary analysis will automatically stop if you begin the run setup steps for a subsequent sequencing run on the MiSeq.
  • Can secondary analysis run on MiSeq while a run is in progress?
    • If a new sequencing run is started on the MiSeq before secondary analysis of a previous run is complete, secondary analysis will be stopped automatically. MiSeq computing resources are dedicated to either sequencing or analysis, and the system is designed in such a way that a sequencing command overrides an analysis command. Secondary analysis can later be requeued from the MiSeq Reporter Analyses tab.
  • Can the ChIP-Seq protocol be used to make libraries from low starting amounts of non-ChIP DNA?
    • Yes, although this requires some adaptation of the protocol. The ChIP-Seq protocol assumes that the input DNA is already fragmented. If the intended starting material is genomic DNA, it will need to be fragmented before use with this protocol. Fragmentation methods such as Covaris offer higher recovery and a tighter size distribution than nebulization, and are preferred for this sort of application. At least 10 ng fragmented DNA should be used.
  • Can the Eco software generate expression fold changes automatically after the assay completes the run, without further command and analysis?
    • The Relative Quantification application calculates and displays fold changes automatically if you defined your reference sample as well as your reference assays. The software automatically calculates and displays error bars for each data point on the graph.

  • Can the Illumina Small RNA v1.5 adapters and primers be purchased separately for directional RNA sample preparation?
    • No, there is currently no Small RNA v1.5 oligo-only kit available. 

  • Can the Infinium HumanMethylation450 BeadChip distinguish 5-hydroxymethylcytosine from 5-methylcytosine?
    • Not at this time. 5-hmC behavior is indistinguishable from standard 5-mC in the Infinium Assay.


  • Can the MiSeq Reporter software be run on a separate PC?
    • MiSeq Reporter can be run on a 64-bit PC with at least 8GB RAM (16–32 GB RAM for optimal performance), Windows Vista or Windows 7, and at least 1 TB of available hard disk space. This allows secondary analysis to be performed offline.
  • Can the MiSeq generate FASTQ files?
    • FASTQ files are generated during secondary analysis by MiSeq Reporter for most analysis workflows. To generate only FASTQ files, specify the GenerateFASTQ workflow in the sample sheet, which generates FASTQ files and then exits secondary analysis.

  • Can the PC Module call genotypes?
    • No, the PC Module performs cluster assignment, but does not call genotypes. This is because the assignment of genotypes polyploid species is highly dependent on the population and biology of the organism. Any downstream genotype assignment should be done with the biology and evolutionary history of the population taken into consideration.

  • Can the TruSeq Amplicon - Cancer panel be sequenced on a HiSeq?
    • The TruSeq Amplicon - Cancer panel is fully supported on the MiSeq. This panel takes advantage of the long read lengths (2 x 150 bp) and the integrated software solution built in to the MiSeq. TruSeq Custom Amplicon and the TruSeq Amplicon – Cancer Panel will be compatible with the HiSeq2500.
  • Can the TruSeq RNA libraries be used with the DSN Normalization protocol?
    • The DSN Normalization protocol was optimized for pre-TruSeq libraries. The TruSeq RNA libraries are not readily amenable to the DSN treatment. There are two primary problems:

      • The Elute-Prime-Fragment mixture is not optimized for non-poly(A) RNA.
      • The PCR primers in TruSeq RNA were optimized for a high yield output. The DSN provides much lower yield due to the duplex-specific nuclease activity, so the PCR primers are not efficient to amplify the DSN-treated TruSeq RNA libraries.
  • Can the TruSeq Sample Prep protocols be run without controls?
    • Yes. To run the protocol without the controls, substitute Resuspension Buffer (RSB) for the control mixture at each step. See the appropriate TruSeq DNA or RNA sample preparation guide for details.

  • Can the TruSeq Small RNA sample libraries be used for directional RNA sequencing?
    • No, not currently. The Directional mRNA-Seq Sample Preparation protocol would need to be optimized to take the longer adapters into account, as the size difference makes adapter dimers more difficult to distinguish from actual inserts using SPRI or column purifications. Illumina encourages customers to continue to use the Illumina Small RNA v1.5 adapters as specified in the Directional mRNA-Seq Sample Preparation Guide .
  • Can the microTUBE AFA Fiber Screw-Cap be used to fragment samples on the Covaris M220?
    • Illumina does not recommend using the microTUBE AFA Fiber Screw-Cap tube (Covaris Part # 520096) with the TruSeq Nano DNA Sample Prep protocol. The solution tends to stick to the sides of the tubes and to the two rods inside the tube, resulting with probable sample loss. Illumina has optimized the M220 shearing conditions using the microTUBE AFA Fiber Pre-Slit Snap-Cap (Covaris Part # 520045) tube and the microTUBE AFA Fiber Crimp-Cap tubes (Covaris Part # 520052).
  • Can the number of PCR cycles be increase for low input samples during TruSeq RNA Access Library Prep?
    • Yes. Illumina has tested 12 PCR cycles with no negative effects. Illumina has not tested > 12 cycles, however it may increase duplicates.
  • Can the number of PCR cycles be reduced with the TruSeq ChIP Sample Prep protocol?
    • The protocol is optimized for 18 cycles of PCR to allow for variability in sample input amounts and quality. High quality samples will likely be able to use a lower number of PCR cycles and still generated adequate amounts of final library. A titration of PCR cycles can be performed to identify the optimal performance for particular samples. Illumina does not recommending increasing the number of PCR cycles as it may skew the representation of the library.

  • Can the wells of a TruSeq HT sample prep adapter plate be reused?
    • No, wells cannot be reused.
  • Can this protocol use purified small RNA as starting material?
    • Yes. Please note that we have seen high sample variability using some commercially available small RNA spin column purification methods, and validation of any purification method may be necessary. If purified small RNA is used as starting material, 10–50 ng should be used.

  • Can thumbnail images be reanalyzed on the HiSeq or HiScanSQ?
    • No. Thumbnail images are for visual inspection only to help diagnose problems with a run. They are not suitable for reanalysis.
  • Can variant classification information be versioned in the Classification Database?
    • No, variant classification and associated notes saved in the Classification Database are not versioned. If the classification category or notes are modified for a given variant in the Classification Database, previous information is overwritten with the current modification.

  • Can we batch process using multiple Eco systems?
    • Currently, batch processing of multiple Eco systems is not supported. Check back periodically and keep an eye on the monthly Illuminotes newsletter for updates.

  • Can we use total-RNA extracted from blood?
    • Yes, total-RNA extracted from blood has been successfully used with this product.
  • Can you explain how tripartite NuZyme probe chemistry works with NuPCR?
    • The key to the NuPCR reaction is the unique NuZyme complex. This three-part probe consists of two PartZyme oligos (PartZyme A and PartZyme B) and universal substrate oligo containing a 5´ fluorophore and a 3´ quencher that binds to the PartZymes. Once the target is amplified via PCR, PartZymes A and B bind to the specific target sequence forming the NuZyme complex. The labeled universal substrate then binds the free substrate region of the NuZyme complex. The inherent catalytic activity of NuZyme cleaves the universal substrate, dissociating the fluorophore from the adjacent quencher, creating detectable fluorescence levels. NuZyme will continue to bind and cleave universal substrate oligos, leading to amplification of the fluorescent signal. Learn more about NuPCR technology .
  • Can you identify tag SNPs for me?
    • Not at this time.
  • Can you tell me about the VeraCode Microbeads?
    • The VeraCode technology is based on cylindrical glass microbeads measuring 240 μm in length × 28 μm in diameter. Illumina uses a proprietary technology to inscribe digital holographic elements within each bead. When a laser beam shines through the bead, the holographic elements diffract the light, creating a code image. Each different bead type contains a unique code that can be used to represent information such as the target of interest in multiplex assays. It can also be used to track critical information, including sample ID, laboratory ID, reagent lots, etc. The high-density codes (24 bit) offer a virtually unlimited number of unique bead types.
  • DesignStudio tells me that there are no designable NuPCR assays in my gene or transcript, what can I do to design an assay for this gene?
    • We recommend selecting a different dye when adding the Target for designing  For example selecting HEX instead of FAM.
  • Did the catalog numbers of the kits that contained MCS3 change when the new MCS4 plus RTE formulation was released?
    • No, all catalog numbers remained the same.
  • Do I get a single long-read from each barcode (384-well) or are there multiple synthetic long reads resulting from a single well?
    • On average, there are several hundred long-read molecules per well. Individual wells produce many long read fragments randomly sampled from across the genome.
  • Do I have to run two flow cells at the same time on a HiSeq 2000?
    • No. You can choose to run only one flow cell at a time.
  • Do I have to use a g-TUBE for TruSeq Synthetic Long-Read DNA library prep if my DNA is already fragmented?
    • The protocol requires starting with high molecular weight DNA. Starting with fragmented DNA or skipping the fragmentation procedure in the protocol will lead to poor results.
  • Do I have to use an x-tracta tool for the excision of the gel band during TruSeq Synthetic Long-Read DNA library prep?
    • The x-tracta has been validated by Illumina, however any gel extraction tool or band excision will work.
  • Do I need a Paired-End Module (PEM) with the HiSeq?
    • No. The HiSeq contains an integrated paired-end fluidics system and a reagent rack for paired-end reagents.
  • Do I need a cBot instrument to be able to run sequencing flow cells on my HiSeq 1500/2500?
    • A cBot is needed for cluster generation on 8-lane High Output flow cells or for loading separate samples in each lane of a 2-lane Rapid flow cell. A cBot is not needed if you only plan to utilize the Rapid run HiSeq functionality and load the same sample across the entire flow cell.
  • Do I need a cBot instrument to run sequencing v4 flow cells on my HiSeq?
    • Yes, a cBot is needed for cluster generation on any eight-lane high output flow cell, including HiSeq v4 and TruSeq v3.
  • Do I need a dedicated server to perform secondary analysis?
    • You can transfer sequencing data from a NextSeq run to BaseSpace for analysis and storage. Additionally, you can configure the NextSeq to transfer data to a local server and perform analysis using third-party software.

  • Do I need a separate computer to run the NextSeq 500?
    • No. The NextSeq contains an onboard computer with instrument control software. All instrument operation is performed from the touch screen on the instrument.
  • Do I need a specific version of the bcl2fastq software package?
    • Yes. To convert zipped BCL files, use bcl2fastq v1.8.4. This version can also convert non-zipped BCL files.
  • Do I need an upgraded MiSeq to run high-plexity samples?
    • You do not need an upgraded MiSeq to run these samples, however, please consider the coverage needed for your study when designing an experiment. An online calculator is provided in DesignStudio to help with these calculations.
  • Do I need any special approval to upload samples to the Illumina iControlDB?
    • Yes, you need to supply an approval letter from your Institutional Review Board (IRB) or equivalent review committee (for international customers) before you begin the upload process. Please see the iControlDB support documents for additional information about the IRB documentation required.

  • Do I need kits, in addition to TruSeq HT sample prep, in order to sequence TruSeq HT dual-indexed libraries?
    • If sequencing TruSeq HT libraries with the HiSeq, HiScanSQ, or GAIIx system on single-read flow cells, the TruSeq Dual Index Sequencing Primer Box, Single Read (single use box) (catalog # FC-121-1003) will need to be ordered separately when using both indices. This add-on box is not required if sequencing a TruSeq HT prepared library with the MiSeq System or on paired-end flow cells for HiSeq, HiScanSQ, GAIIx. Additional SBS reagents may be necessary to account for all index cycles.

  • Do I need new license keys for modules that I do not already own?
    • Yes. If you do not already own the module, you need to purchase new license key(s).
  • Do I need oil to load a flow cell on the HiSeq or HiScanSQ?
    • No. The HiSeq and HiScanSQ do not require immersion oil to properly load a flow cell in the way that the Genome Analyzer does. Instead, the flow cell is held in place by a vacuum, which removes air and replaces the need for immersion oil.
  • Do I need to begin clustering with one of the two default algorithms, OPTICS or DBSCAN?
    • No, you can also cluster by defining #clusters, if known based on the biology of your samples.
  • Do I need to cluster all SNPs in the data set by the same algorithm and clustering options?
    • No, you can use different algorithms and clustering options for different SNPs. The goal is to find optimal parameters for each SNP matching the biology of your samples.

  • Do I need to dilute my NuPCR Assays prior to use?
    • NuPCR assays come supplied as 20X concentrated stock solution.  Simply dilute 1:20 into your final PCR reaction volume. For example for a 20 µl reaction, add 1 µl of NuPCR Assay.  
  • Do I need to have a copy of BeadStudio to upload data to the Illumina iControlDB?
    • Yes, BeadStudio v3.1 or higher is required to upload data to the database.
  • Do I need to make full 96-plex or 24-plex pools?
    • No, lower levels of multiplexing are possible. Please see the Nextera DNA Sample Preparation Guide for more information on pooling recommendations for samples less than the full kit size.
  • Do I need to normalize my data for TruSeq Targeted RNA Expression?
    • Yes, you will need to select internal normalizer genes from RNA-Seq or Microarray data from samples similar to those that will be run on the assay. The genes should be invariant across the samples being tested. Assays specific to the normalizer genes need to be included in the final TOP panel.
  • Do I need to order the robot upgrade to automate the GoldenGate Assay for Methylation?
    • Automation for the GoldenGate Assay for Methylation is processed using a new robot control application bundled into the GTS robot control software. Please contact Technical Support at techsupport@illumina.com for the latest version of robot control software.
  • Do I need to perform indexing if preparing samples with Nextera?
    • No, you can run single samples without indexing. However, you must order one of the two available Index Kits in order to perform the PCR required for the library prep: either the 24 Index Kit with sufficient reagents for 96 samples (Catalog # FC-121-1011) or the 96 Index Kit with sufficient reagents for 384 samples (Catalog # FC-121-1012). When clustering/sequencing these samples on cBot/Cluster Station and HiSeq/HiScanSQ/GAIIx, even if you do not perform indexing (single or dual), you will need to use the new TruSeq Dual Index Sequencing Primer Kits (Single Read Kit is Catalog # FC-121-1003 and the Paired End Kit is Catalog # PE-121-1003).
  • Do I need to prime SBS reagents in HiSeq v4 run mode?
    • Yes. Priming SBS reagents is required. HCS prompts you for this step, which takes about 15 minutes.
  • Do I need to prime SBS reagents in TruSeq Rapid mode?
    • No. Priming is integrated into the HCS workflow for TruSeq Rapid runs.
  • Do I need to purchase AMPure beads if using the TruSeq DNA PCR-Free Sample Prep kit?
    • No, TruSeq DNA PCR-Free sample prep kits contain Sample Purification Beads (SPB) that are used for size selection and clean-up steps. They do not require the user to supply AMPure beads separately.
  • Do I need to purchase AMPure beads if using the TruSeq Nano DNA Sample Prep Kit?
    • No, the kit contains Sample Purification Beads (SPB) that are used for size selection and clean-up steps. They do not require the user to supply AMPure beads separately.
  • Do I need to purchase Nextera sequencing primers separately?
    • The correct primers are already included with MiSeq cartridges. However, the Nextera sequencing primers will need to be purchased separately when sequencing libraries on HiSeq2000, HiSeq 1000, HiScanSQ, or GAIIx. These primers are included in the TruSeq Dual Index Sequencing Primer Kits: the Single Read Kit is Catalog # FC-121-1003 and the Paired End Kit is Catalog # PE-121-1003.
  • Do I need to purchase the dual index primer reagent box for sequencing Nextera Rapid Capture Enrichment libraries?
    • The TruSeq Dual Index Sequencing Primer Kit (catalog #PE-121-1003) is required for clustering libraries on the cBot and paired-end sequencing on the HiSeq 2000, GAIIx, and in High Output mode on the HiSeq 2500. This kit is good for one run and contains the required primers for dual index sequencing (HP10, HP11, HP12).

      The dual index primers (HP10, HP11, HP12) are included in MiSeq reagent kits and HiSeq 2500 Rapid reagent kits and do not need to be purchased separately for these types of runs.

  • Do I need to re-titrate my samples for use with flow cell v3?
    • Yes. The new cluster protocol and wider lanes on flow cell v3 make it imperative to re-titrate your samples to optimize cluster densities for higher throughput. qPCR is highly recommended.

  • Do I need to remove the ribosomal RNA prior to labeling?
    • It should not be necessary to remove rRNA before labeling. The ribosomal RNA does not get amplified in the protocol and should represent a very small percentage of the final product.
  • Do I need to specify a genome folder in my sample sheet for the metagenomics workflow?
    • No reference genome is necessary for the MiSeq metagenomics workflow.

  • Do I need to use a cBot to cluster a HiSeq/HiScanSQ flow cell?
    • Yes. The HiSeq flow cell, also used on the HiScanSQ, requires the use of a cBot for clustering on the flow cell prior to sequencing.
  • Do I need to use a cBot with the MiSeq?
    • No. Cluster generation is performed on the MiSeq instrument as part of the run. No other instruments are required for sequencing on the MiSeq.

  • Do I use the same library input concentration for a HiSeq 2500 rapid run flow cell as for a high output flow cell?
    • Cluster density tends to be about 15% higher on rapid run two-lane flow cells compared to high output eight-lane flow cells when loaded with the same concentration of library. Take this 15% difference into account when targeting cluster density on your HiSeq 1500 and 2500 systems.
  • Do I use the same library input concentration for a HiSeq v4 flow cell as for a TruSeq v3 High Output flow cell?
    • To achieve the increased cluster density using the HiSeq v4 configuation, the loading concentration of library has to be increased. For most standard libraries, a 50% greater concentration is sufficient. For more information, see Denaturing and Diluting Libraries for the HiSeq and GAIIx (part # 15050107) .
  • Do NextSeq 500 reagents use the same SBS chemistry as the HiSeq and MiSeq reagents?
    • All Illumina sequencers use the Illumina patented reversible-terminator SBS chemistry. The NextSeq 500 System employs the latest evolution in SBS technology: a novel two-channel SBS method that supports reduced cycle time and data processing time.
  • Do TruSeq HT dual-indexed libraries still sequence 1 bp more than the index sequence used for demultiplexing?
    • No. Dual-indexed libraries sequence eight bases of actual index sequence rather than six bases plus one more for phasing calculations. For short sequences there are no phasing/prephasing corrections, so it is not necessary to sequence one additional cycle.

  • Do TruSeq Stranded Total Stranded Sample Prep Ribo-Zero kits only remove ribosomal RNA?
    • TruSeq Stranded Total RNA Sample Prep kits remove ribosomal RNA only. The Human/Mouse/Rat kits remove cytoplasmic ribosomal RNA and the Ribo-Zero Gold kits remove cytoplasmic plus mitochondrial ribosomal RNA.

  • Do any manufacturers provide well plates that can be used in the Eco system?
    • At this time, Illumina is the only provider of Eco plates.
  • Do homopolymers and repetitive DNA regions impact sequencing efficiency?
    • Homopolymers do not impact sequencing. The number of uniquely alignable reads is a function of the repeat content, so this will have an impact on productivity. With longer reads and paired-end sequencing, this may be less of an issue.
  • Do probe/Taqman master mixes work with the NuPCR assay?
    • No, NuPCR is optimized for use with the NuPCR Master Mix.
  • Do the TruSeq Stranded Total RNA Sample Prep Kits with Ribo-Zero Globin or Plant work on degraded RNA?
    • All Ribo-Zero kits are compatible with degraded RNA. For more information, see the TruSeq Stranded Total RNA Sample Preparation Guide .
  • Do we need to upgrade our servers for analysis of HiSeq data?
    • You may need to upgrade your computing infrastructure to accommodate the new data output rate (Gigabases/hr) of the HiSeq 1500/2500 in Rapid Run mode, which is roughly twice the data output rate of a High Output run. However, Illumina offers two new methods to reduce the data output rate by more than 50% without affecting data quality, allowing you to leverage your existing infrastructure to store either twice the amount of data with the same amount of storage (High Output mode) or to keep the Rapid Run mode data output rate the same as your HiSeq 1000/2000 data output rate. The recommended configuration for one HiSeq is an IlluminaCompute Standard appliance. More information is available here or from your account manager.
  • Do you consider underlying polymorphisms in the Design Score calculation?
    • The Design Score reflects underlying polymorphisms based on the extent that the polymorphisms are annotated in dbSNP. If you have additional annotation information regarding polymorphisms around SNPs of interest, you should consider submitting these SNPs in a sequence list to be sure this information is taken into account.
  • Do you have any published references which have used NuPCR?
    • Not at this time.  We will post citations to illumina.com as we become aware of them.
  • Do you have protocols available for whole-genome or reduced-representation bisulfite sequencing?
  • Do you optimize the NuPCR master mix for different instruments?
    • NuPCR Master Mix and PCR cycling parameters are  optimized for use on many qPCR instruments.
  • Do you provide detailed protocol for setting up NuPCR reactions on different instrument/software?
    • For NuPCR one set of conditions applies to all machines. Refer to the NuPCR Assay Protocol Guide for details on the cycling parameters.
  • Do you recommend a bisulfite conversion kit?
    • Illumina recommends the Qiagen EpiTech® Bisulfite Kit, PN 59104.
  • Do you recommend an RNA purification protocol?
    • Illumina does not recommend a specific RNA purification product. However, any product that yields pure, intact RNA of good quality that retains (at least) most of the small RNAs should work well with our miRNA assay. We have generated good data with RNAs extracted with the Ambion kit, Qiagen kit, Trizol, etc. NOTE: For any given study, it is ideal to isolate the RNAs using a single method.
  • Do you support an automation option for custom Infinium?
    • Yes, both manual and automated product offerings are available.
  • Does BaseSpace require a sample sheet?
    • If you choose to use BaseSpace for run monitoring only, a sample sheet is not required. If you want to use BaseSpace for data storage and analysis, a sample sheet is required. The sample sheet must be in HiSeq Analysis Software format, and all samples listed in BaseSpace must be indexed OR all must be non-indexed. Mixing indexed and non-indexed samples is not possible on a flow cell when using BaseSpace.
  • Does DNA require quantitation after bisulfite conversion?
    • No. However, the user guide provides directions for quantifying the DNA before bisulfite conversion.
  • Does GenomeStudio software currently offer features for small RNA Sequencing and Tag Sequencing data analysis and visualization?
    • Not at this time.
  • Does Illumina VariantStudio support TruSight Tumor data?
    • Yes. The Amplicon-DS workflow in the MiSeq Reporter software is used for the alignment and variant calling of TruSight Tumor data. The resulting VCF files can be imported into VariantStudio. Illumina recommends that you use the merged VCF file from the Amplicon-DS workflow. In these files, the variant call information from both the forward pool and reverse pool has been merged.

  • Does Illumina VariantStudio support the genome VCF (gVCF) format?
    • Yes. VariantStudio v2.1 and v2.2 support VCF and gVCF file formats. For more information, see the VariantStudio User Guide.

  • Does Illumina curate the annotation database?
    • No. Illumina provides annotations from the respective annotation sources as is.

  • Does Illumina have any recommendation about ChIP methods for TruSeq ChIP sample prep?
    • Illumina does not provide any recommendations as methods for ChIP pulldown and fragmentation are dependent upon individual antibodies and procedures. Please reference literature or other sources for recommendations.

  • Does Illumina offer TruSeq Sample Prep Oligo Only Kits?
    • Oligo Only Kits are not offered for the TruSeq DNA and RNA Sample Prep kits.
  • Does Illumina offer a custom targeted resequencing product?
  • Does Illumina provide annotation (gene, distance) for the rsSNPs?
    • Yes, annotation for rsSNPs is available upon request.
  • Does Illumina provide antibodies for ChIP assays?
    • No, Illumina does not provide antibodies.


  • Does Illumina provide classified variants in the Classification Database?
    • No, Illumina does not provide any classified variants. VariantStudio provides an empty Classification Database that you can populate by uploading classified variants from an external source or by manually classifying variants in samples that are being analyzed in VariantStudio.

  • Does Illumina provide nebulizers in the TruSeq DNA Sample Prep kits?
    • Nebulizers are not provided, but can be purchased separately through iCom.
  • Does Illumina recommend a specific kit for bisulfite conversion?
    • EZ DNA Methylation Kit for 50 DNA reactions (supplied with capped columns) (Zymo Research, catalog # D5001), EZ DNA Methylation Kit for 200 DNA reactions (supplied with capped columns) (Zymo Research, catalog # D5002), EZ-96 DNA Methylation Kit for 2x96 DNA conversion reactions (deep-well Zymo-Spin I-96 Filter Plate) (Zymo Research, catalog # D5004) Any other methylation kits are not recommended for use with this assay.
  • Does Illumina recommend any the commercially available methylation standard DNA: methylated, hemimethylated, or unmethylated?
    • Illumina has not extensively tested any of the commercially available standards, and does not recommend any of them at this time. In house, Illumina has used standards generated in our own laboratory. Our methods for generating the standards are described in the following article:

      Bibikova M, Le J, Barnes B, Saedinia-Melnyk S, Zhou L, et al. (2009) Genome-wide DNA methylation profiling using Infinium assay, Epigenomics 1:177-200

  • Does Illumina recommend background subtraction for analysis?
    • Background subtraction is required when you compare data run on different types of scanners because of technical disparities: for example, the iScan and HiScan have very different offsets. Background subtraction has a much smaller effect when you scan chips on the same scanner, and might not be necessary. We suggest that you analyze a subset of data with and without subtraction, and choose the one you prefer based on your results.

  • Does Illumina recommend filters or cutoffs to use for different scenarios?
    • No. Filtering parameters vary depending on the application and objective of the investigator.

  • Does Illumina recommend requantitation of DNA samples after bisulfite conversion?
    • Requantitation is not necessary as long as you use at least 500–1000 ng DNA for the bisulfite conversion. It is critical to accurately quantitate the input DNA concentration with PicoGreen to ensure that you add sufficient DNA to the bisulfite conversion reaction. Bisulfite conversion renders DNA less complementary, so much of the DNA will be denatured and be more difficult to quantitate accurately.

  • Does Illumina recommend specific bisulfite conversion kits?
    • Yes. Illumina recommends the following of-the-shelf bisulfite conversion kits from Zymo Research.

      Catalog Number

      D5001    50 reactions (single-column format)

       

      D5002    200 reactions (single-column format)

       

      D5004    2×96 reactions EZ-96 DNA methylation kit (deep-well format)
  • Does Illumina recommend using the TruSeq Stranded Total RNA Sample Prep Kit with Ribo-Zero Globin on any sample other than blood?
    • The Ribo-Zero Globin kit has been specifically tailored for blood samples from human, mouse, or rat. In addition to removing cytoplasmic and mitochondrial rRNA, it will also deplete globin mRNA. Illumina recommends using the Ribo-Zero Gold kit for samples other than blood.
  • Does Illumina sell Real-Time PCR reagents?
    • Yes. NuPCR reagents for qPCR provide a specific and sensitive solution for analyzing gene expression using real-time PCR. NuPCR reagents run on all real-time PCR instruments. See NuPCR for more information. Use Illumina's web-based tool DesignStudio to design NuPCR assays and primer sets for use with DNA binding dyes. See DesignStudio for more information.
  • Does Illumina supply automated protocols for use with their gene expression products?
    • Automation of the DASL® Assay is available. However, Illumina currently does not offer automation for the Direct Hyb protocol. Contact Ambion for information about automation of the TotalPrep protocol.

  • Does Isaac utilize quality information during SNP-calling?
    • Base qualities are considered during mapping to calculate the alignment score and during SNP and indel calling to calculate the variant quality scores.
  • Does MiSeq Reporter recognize *.fasta or *.fa?
    • Yes, MiSeq Reporter recognizes both extensions.
  • Does MiSeq require an uninterruptible power supply (UPS)?
    • The use of a UPS is optional. However, a UPS is highly recommended to protect the instrument in the case of a power surge. For more information, see the MiSeq System Site Prep Guide.

  • Does TruSeq Custom Amplicon use the same PCR/sequencing primers as Nextera?
    • No. The two assays use different oligos.
  • Does TruSeq RNA Sample Prep provide information about the originating strand from which the DNA was transcribed?
    • In the random priming process to generate cDNA with the best coverage, this information is not retained. If originating strand information is required, please refer to the Directional mRNA-Seq Sample Preparation Guide or other published applications.

  • Does TruSeq Synthetic Long-Read DNA data analysis require BaseSpace?
    • Yes, sequencing runs must be streamed to BaseSpace to utilize the corresponding TruSeq Long-Read Assembly and TruSeq Phasing Analysis apps on BaseSpace for analysis.
  • Does TruSight DNA Amplicon use the same PCR/sequencing primers as TruSeq Custom Amplicon or Nextera?
    • TruSight DNA Amplicon uses the same PCR/sequencing primers as TruSeq Custom Amplicon. Nextera uses different oligos.
  • Does VariantStudio perform variant classification?
    • No, you perform variant classification. With VariantStudio, you can provide annotations that can be used to classify variants within VariantStudio. After you assign a classification category to a variant, the information is saved to the Classification Database so that it can be applied to the same variant observed in other samples.  Classifications saved in the Classification Database can be applied automatically to other samples through the Apply Classifications from Database menu.

  • Does VariantStudio require an internet connection?
    • Yes, Illumina VariantStudio requires internet connection for annotating variants because the annotation database, known as the Illumina Annotation Service, resides in Amazon Web Services. After variants are annotated and saved in a project, an internet connection is not necessary.

  • Does VariantStudio run on a Mac or Linux system?
    • No. VariantStudio is tested and supported on Windows 7 or later. Virtualization software like Parallels or VMware lets you run a Windows application on a Mac or Linux. Therefore, it is possible to run Illumina VariantStudio through Parallels on a Mac. However, this method is an unsupported function. It is recommended that you give your VM at least 2 GB of RAM and close any other programs.

  • Does VariantStudio support analysis of structural variations?
    • No. VariantStudio does not currently support copy number variations (CNVs) or structural variations (SVs).
  • Does a HiSeq 1500 or HiSeq 2000 need to be upgraded to a HiSeq 2500 to run the HiSeq v4 chemistry?
    • No. All eligible HiSeq 1500 and HiSeq 2000 systems only need the FPGA and software upgrades to use the HiSeq v4 reagents.
  • Does cDNA cluster in the same way as gDNA?
    • cDNA libraries should be considered an equivalent to gDNA for clustering and sequencing steps, assuming an equivalent complexity of the sample.

  • Does custom Infinium support Tri/Tetra-allelic SNP assays?
    • Not at this time.
  • Does iControlDB generate different report types other than the matrix format, forward strand report, because my Final Case Report is not in the forward strand orientation?
    • No, not at this time.
  • Does the Eco Real-Time PCR system require separate software for analysis?
    • No. In addition to instrument control and data collection capabilities, the Eco Real-Time PCR System comes with the EcoStudy data analysis software. If more advanced analysis is required, you can export all raw data in Excel or CSV format for convenient analysis in other third-party software.

  • Does the Eco software have individual profiles for users with password protection, file storage, and data analysis preference?
    • The Eco software was designed for individual users, so it does not provide user profiles or password protection. However, you can configure Windows 7 on the provided computer for multiple users, giving each user an individual login with personalized settings and folders.
  • Does the HiSeq Analysis Software replace CASAVA?
    • No, the HiSeq Analysis Software provides analysis of libraries prepared with the Nextera Rapid Capture exome enrichment kit and human Whole Genome Sequencing using only the hg19 genome as a reference. CASAVA provides analysis of additional applications such as RNA sequencing, Exome sequencing, targeted resequencing, and Whole Genome Sequencing using a more extended set of reference genomes available on Illumina.com's iGenomes page. (http://support.illumina.com/sequencing/sequencing_software/igenome.ilmn). More information on CASAVA is available here: http://www.illumina.com/software/genome_analyzer_software.ilmn#casava

  • Does the HiSeq require an uninterruptible power supply (UPS)?
    • Illumina provides a region-specific UPS with the HiSeq instrument.
  • Does the Library QC workflow in MiSeq Reporter perform alignment?
    • Yes, alignment is performed in the Library QC workflow; however, it is performed using a faster and less sensitive setting, which provides a much faster turnaround time. Variant calling is not employed for this workflow.
  • Does the MiSeq have an instrument PC?
    • Yes. The MiSeq includes a fully-integrated instrument computer that runs on Windows 7 Embedded and contains the operating software, control software, and analysis software.

  • Does the MiSeq reagent cartridge contain sequencing primers for all applications?
    • Yes. The MiSeq reagent cartridge contains sequencing primers for TruSeq DNA and RNA samples, TruSeq Small RNA samples, TruSeq Custom Amplicon libraries, and Nextera DNA samples.
  • Does the NextSeq 500 perform secondary analysis on the instrument?
    • The data set generated by the NextSeq is too large for on-instrument analysis. Data must be transferred to BaseSpace or a local server for secondary analysis.
  • Does the TruSeq Stranded Total RNA Sample Prep Kit with Ribo-Zero Globin subtract Globin from non-human RNA?
    • The Ribo-Zero Globin kit supports human, mouse, and rat. Use of this kits with other organisms has not been tested and is not supported.

      Please contact Illumina technical support with the rRNA sequence for your organism of choice to determine if your organism is compatible with the Ribo-Zero Globin kit. Please note that the analysis will be done in silico and does not guarantee rRNA removal.

  • Does the TruSeq Stranded Total RNA Sample Prep Kit with Ribo-Zero Plant kit work on plant leaves only, or is it also compatible with seeds and roots?
    • The Ribo-Zero Plant kit contains oligos that will remove cytoplasmic and chloroplast rRNA from leaves, seeds, and roots.
  • Does the TruSeq Stranded Total RNA Sample Prep Kit with Ribo-Zero Plant work on all plant species? What about algae?
    • The Ribo-Zero Plant kit will remove rRNA from a broad variety of bacteria, fungi/yeast, plants, and eukaryotic total RNA samples. It has been laboratory tested against Arabidopsis, maize, wheat, rice, corn, and soy bean. In silico testing has also demonstrated compatibility with Japanese honeysuckle (Lonicera japonica), mirror bush (Coprosma repens), moss (Physcomitrella), Tomato (Solanum lycopersicum), Vietnamese coriander (Persicaria odorata), and water clover (Marsilea vistita).

      Please contact Illumina technical support with the rRNA sequence for your organism of choice to determine if your organism is compatible with the Ribo-Zero Plant kit. Please note that the analysis will be done in silico and does not guarantee rRNA removal.

  • Does the TruSight Myeloid Sequencing Panel detect partial or tandem duplicates?
    • MiSeq Reporter does not detect duplication events. Reads from this type of rearrangement are not expected to align and will likely be placed in the Unaligned folder by MiSeq Reporter.
  • Does the TruSight Myeloid Sequencing Panel detect translocations?
    • No, this panel is designed for the detection of SNVs and small insertions and deletions only. Translocations will not be detected with this assay.
  • Does the fluorescent acquisition occur through the side or top of the plate?
    • The acquisition is done by a CCD camera through the top of the plate and through the optical adhesive sealer.

  • Does the install of the PC Module require a license key?
    • No, the PC module is a standalone software which does not require a license key.
  • Does the mRNA-Seq kit include controls?
    • No specific controls are included with this kit. The PhiX sequencing control (sold separately) is necessary for calibrating the instrument for each run. For a sample-prep-level positive control, we recommend the Universal Human Reference RNA from Strategene.
  • Does the software allow the user to perform a regular check on the performance of the Eco detection system?
    • Use the Eco evaluation plate to test the performance of the instrument whenever needed.

  • Does this protocol offer strand-specific information?
    • This protocol is designed for quantitative shotgun cDNA sequencing. Therefore, you get reads from both ends of the cDNA fragments and not from a specific strand. We determined that this protocol is the best method available for creating even, unbiased coverage of the entire length of the cDNA molecule. Our scientists tested many methods and found that this feature of the protocol provides the most quantitative and reproducible results for counting transcripts. If you are interested in generating strand-specific information, several publications have demonstrated this capability on the Genome Analyzer: for example, Lister et al., Highly Integrated Single-Base Resolution Maps of the Epigenome in Arabidopsis, Cell (2008), doi:10.1016/j.cell.2008.03.029.
  • Exactly what files are uploaded as instrument health data?
    • The files uploaded as instrument health data are RunInfo.xml, RunParameters.xml, RTAComplete.txt, InterOp files, and RTAConfiguration.xml.
  • For custom Infinium can I assay SNPs that are next to each other (i.e., < 60 bp apart)?
    • Yes. In the Infinium Assay, the effect of underlying polymorphisms is not critical to overall performance.
  • For custom Infinium content, what is the expected overall conversion to functional assays?
    • The conversion rate depends on many factors including our stringent QC criteria during manufacturing, the sequence nature of the chosen SNPs, and criteria used for Gentraining. To maximize chances of success, we recommend selecting validated SNPs and/or SNPs with high design scores. In general, we expect the final Design Conversion Rate to average 80%.
  • For dual-indexed libraries, how many cycles are performed for index reads?
    • Dual-indexed runs on the HiSeq comprise 8 bp of index sequence rather than 6 bp plus a seventh for phasing calculations.
  • For publication purposes, how should I cite that I have uploaded or downloaded samples from iControlDB?
    • Please reference the Illumina iControlDB web page http://www.illumina.com/science/icontroldb.ilmn and/or Illumina, Inc., San Diego, CA, whichever is the preferred format of the journal to which you are submitting.

  • For the bisulfite conversion, do I need specific hardware?
    • We recommend performing this reaction in a thermocycler in order to keep the temperature stable. Any PCR machine can be used for the bisulfite conversion reactions, but this reaction MUST be done in the pre-PCR room.
  • Has the tile layout or numbering changed due the wider lanes on Flow Cell v3?
    • No, the tile numbering is unchanged from the format introduced in HCS v1.3. However, when using Flow Cell v3, the tile numbering reflects the three-swath imaging pattern, where a 3 in the tile number represents the third swath.

  • Have Illumina’s gene probes been mapped to any public database?
    • Yes. Our probes (human and mouse) have been mapped to Ensembl. The Illumina probe track can be turned on by checking the box next to Illumina Probes on the DAS Sources dropdown menu in Contigview. When the page refreshes, blue features indicate the location of probes. Clicking on a probe opens up a floating menu with probe information and a link to a page giving more information about the probe. The GFF text files giving the mapping information for probes to mouse and human can be downloaded from the Sanger Institute website ( http://www.sanger.ac.uk/Software/ formats/GFF/): - Human GFF file (right-click and select Save As) - Mouse GFF file (right-click and select Save As) More information about the GFF data format with an explanation of the fields can be found at: http://www.sanger.ac.uk/Software/formats/GFF/

  • Have different levels of DNA fragmentation been tested with this kit?
    • Customers can use the Illumina FFPE QC Kit (WG-321-1001) to determine whether their samples meet the minimum quality criteria for inclusion in a Cancer Panel experiment. This protocol is qPCR based, where a Ct (threshold cycle) cut-off allows users to determine whether their samples will succeed in the TruSeq Amplicon assay or not. This measurement represents how well DNA amplifies and can be used to infer DNA quality. Testing has demonstrated that over 80% of samples passing the Illumina FFPE QC Kit protocol are expected to succeed in the TruSeq Amplicon assay.
  • How am I notified when there is a new version of the Illumina Annotation Service?
    • If a new version of the Illumina Annotation Service is released at the same time as a new software version release, the updates to the annotation database are specified in the software release notes. Otherwise, you receive an email notification informing you that a new database version is available. The email outlines the changes made relative to the previous annotation database version.

  • How are ChIP-Seq runs analyzed with Illumina software?
    • ChIP-Seq runs should be aligned using the eland extended module of Gerald in CASAVA1.7. CASAVA 1.8 is not compatible with ChIP-Seq analysis. In CASAVA1.7, the Gerald config.txt file should include the line "WITH_SORTED true" to generate the sorted.txt files for each lane. CASAVA itself does not need to be run for ChIP-Seq runs. The GenomeStudio ChIP-Seq module requires the sorted.txt files and Summary.htm from Gerald.

  • How are counts normalized in the RNA Sequencing Module?
  • How are images taken on the HiSeq or HiScanSQ?
    • Images are taken using a time delayed integration (TDI) line scanning optical system with four CCD sensors. The TDI line scanning system greatly increases throughput by maximizing camera utilization.
  • How are methylation levels measured by the Infinium HumanMethylation450 BeadChip?
    • A beta value (β) is calculated for each CpG locus.

  • How are the HiSeq X HD Reagent kits configured?
    • Each kit includes two flow cells and all required reagents, including clustering, SBS, paired-end, and indexing reagents. One kit provides consumables for a 150-cycle paired-end sequencing run plus indexing on both flow cell A and flow cell B.

  • How are the VeraCode Carboxyl Beads used?
    • Carboxyl beads have a carboxylated surface, enabling development of protein-based assays. To achieve multiplexing, pool together different tubes of uniquely coded carboxyl beads after immobilizing with proteins of interest.
  • How are the VeraCode Universal Beads used?
    • Each uniquely coded VeraCode Universal Bead has a unique oligonucleotide capture sequence attached and can be used to design nucleic-acid based assays. For example, to develop a 3-plex reaction using a single-color detection assay, such as Allele Specific Primer Extension (ASPE), pool together six different tubes of unique VeraCode Universal Oligo Beads (one bead type per allele).
  • How can I be sure we are targeting only miRNA in the total-RNA?
    • This is achieved by two-step discrimination (1) sequence hybridization - the specificity of the miRNA specific probe which targets the pre and mature miRNA species, and (2) enzymatic primer extension - to enhance the discrimination between members of miRNA families and between miRNA and other similar sequences in the total-RNA (e.g., mRNA targets). Illumina has obtained very similar expression profiles with total-RNA and enriched small RNA species, suggesting that cross-hybridization (if any) from the total-RNA is minimal.
  • How can I calculate a p-value from the DiffScore?
    • p = 10'(DiffScore*sgn(µcond-µref)/10)

  • How can I check whether the copying of files proceeds fast enough?
    • Do the following to ensure that your network is performing fast enough for a particular run:

      1. Open the copylog.txt file located in \Data. Each entry has the following format:
        7/1/2009,07:01:31.843,0,0,0,Copy D:\Runs\090630_HWUSI-EAS713_97772299_42FRP\Processed\L008\C13.1\s_8_65.cif to \\smb11\smb\sata825\production\090630_HWUSI-EAS713_97772299_42FRP\Data\Intensities\L008\C13.1\s_8_65.cif,406,0,0
      2. Look for entries designated Copy (as shown in step 1) after the first series of numbers in the entry. These entries describe copy events.
      3. Collect the file name and copy time. The third number from the end specifies the time (in ms) that it took to copy the file to its destination.
      4. Go to the destination folder and look up the corresponding file size.
      5. Divide the file size by the copy time, which gives you the copy speed for that file. For a typical image file (7.5 Mbyte), in this example the copy speed is 18.5 Mbyte/second.

      6. Repeat steps 2-5 for a few more files. The typical copy speed should be at least 10 Mbyte/s, ideally 15 Mbyte/s or higher.

      7. If the copy speed is consistently significantly less than 10 Mbyte/s or lower, your network does not perform fast enough. 

      See the FAQ “What should I do if the copy speed is too slow?” for suggested solutions.

  • How can I combine two regions in close proximity into one larger region?
    • When you create a merged table, you can specify how close two regions must be in number of base pairs.
  • How can I compensate for samples that have variable concentrations, quality, or integrity?
    • This situation is a challenge for any system trying to quantify gene expression levels. Variable quality is more challenging than variable compensations for the mRNA-Seq Assay. As described in the question about RNA degradation, we recommend against comparing results across samples that have a large range of quality and integrity, because there will be a large effect on the read coverage across the full length of the transcripts.

      If your RNA samples are of distinctly different quality, start by re-purifying all of the lesser-quality samples with the goal of having the highest quality RNA possible. Next, check that the samples you want to compare are of similar quality and quantity. We do not yet have analytical methods to normalize for these effects in mRNA-Seq data.

  • How can I create subpanels for use in VariantStudio?
    • Subpanels can be created by generating gene lists in HGNC nomenclature for the regions of interest. The complete TruSight One gene list can be found at (www.illumina.com/products/trusight-one-sequencing-panel.ilmn ) for reference. Upload this subpanel gene list when importing *.vcf files into VariantStudio to filter variants based on the shortened gene list.
  • How can I get GenomeStudio software?
    • If you are a new user, you can download GenomeStudio software from iCom, or you can request it from your Regional Account Manager, Regional Sales Manager, or Illumina Technical Support. If you are a current GenomeStudio software user, you can download the latest version of GenomeStudio software from the GenomeStudio Portal.
  • How can I get a full list of the genes covered by the Cancer Panel?
    • The manifest file for the TruSeq Amplicon - Cancer Panel is available for download from MyIllumina.
  • How can I get copies of the user and service documentation?
    • Hard copies of the setup poster and user guide are included with each Eco system, and a soft copy is available on the USB drive. You can also download PDFs of the Eco documentation from http://www.illumina.com/ecoqpcr.

      Illumina will handle all service and repair requests. If you have a service or repair request, please contact Technical Support.

  • How can I improve the designability and coverage in my TSCA DesignStudio Project?
      1. Increasing the size of the target to design against can rescue previously ‘undesignable’ regions. The increased size of a target gives DesignStudio a little more flexibility to fit a higher scoring amplicon over the desired target bases.
      2. Enable the ‘SNP OFF’ feature – With this option, DesignStudio will be more aggressive when placing amplicons by putting less importance of the location of SNPs in the design process. This can help rescue ‘undesignable’ regions if DesignStudio avoided regions with low frequency or poorly annotated SNPs in the database, leading to low designability. DesignStudio uses dbSNP 131 (human hg19), snp 128 (mouse mm9), snp 125 (rat rn4) and Ensembl UMD3.1 snps (bovine) to position probes that avoid SNP and indel locations to improve accuracy and performance of the design for that region.
      3. Change the context of the panel – for example, putting highly homologous target sequence into the same multiplex design can be problematic for designing probes to discretely amplify each target. Moving problematic regions into a separate design can frequently improve the designability.
  • How can I obtain KaryoStudio?
    • Contact your account manager for information about getting new versions of KaryoStudio software.
  • How can I obtain new versions of cnvPartition?
  • How can I optimize cluster formation?
    • Short fragments tend to create smaller clusters allowing greater data density. The optimal fragment size for single-read run is 150–300 bp. The optimal fragment size for paired-end run is 250–500 bp.
  • How can I prevent or reduce amplification in my no-template control (NTC) reactions from SYBR Green I assays?
    • Amplification in NTC reactions can either be from contamination or non-specific amplification. Performing melt curve analysis can help identify if the signal is from contamination or from non-specific amplification.

      If it is contamination, the melting curve of the NTC reaction will have the same Tm as your target sequence. Good aseptic technique, using aerosol-resistant pipette tips and a Real-Time PCR master mix with dUTP and UDG, can help to reduce any potential contamination.

      If the signal is due to non-specific amplification, the melting curve of the NTC reaction will have a different Tm than the target sequence. The most common type of non-specific amplification is primer-dimer formation, and there are a number of ways to reduce this.

      Optimal primer design is an important first step in preventing primer-dimer formation.

      • Primer pairs should be screened using oligonucleotide analysis tools for any secondary structure, self-annealing, or cross-annealing.
      • A hot-start DNA polymerase will prevent the extension of non-specific primer annealing during the reaction setup.
      • Decreasing the primer and Mg++ concentrations can reduce primer-dimer formation, but can have a negative effect on amplification efficiency.
      • Additives such as glycerol and DMSO have been known to reduce primer-dimer formation.

      If these steps do not help reduce primer-dimer formation, redesigning the primers is necessary to obtain good results.

  • How can I share my Nextera Rapid Capture Custom Enrichment design content with my collaborator?
    • Custom target designs can be shared by saving Target Regions from your DesignStudio project to a .csv file using the Export function. The exported .csv file can be sent to your collaborator and imported into a new DesignStudio project, using the file upload method, to view the Target Regions of interest. Probe sequences can also be shared using the Export function of the Probes table.
  • How can I take data from samples that have been run in the past and see if those same found regions appear in samples that are run in the future?
    • You can adjust the information contained within the known regions report. Refer to the user guide, which describe how to customize the known regions table so you can track your favorite regions in the additional samples.
  • How can I tell if my genes of interest are present in the TruSight One Sequencing Panel kit?
    • The TruSight One *.bed file details all regions of interest (ROI) and lists all targets of enrichment for this assay. Filter by gene name (HGNC nomenclature), or reference the coordinates of your loci of interest in the human genome reference (hg19 build). The gene list is also available on the product page (www.illumina.com/products/trusight-one-sequencing-panel.ilmn ).
  • How can I tell which genome my sequences were aligned to?
    • See the CASAVA User Guide, available in the GenomeStudio Portal.
  • How can I visualize my results and compare them to GEX data?
    • miRNA data can be combined with mRNA gene expression BeadStudio projects (Whole genome or DASL) in BeadStudio Gene Expression v3.2 or later. One tool to help visualize positive and negative correlations is hierarchical clustering w/ absolute correlation.
  • How can I zoom into a specific region of interest?
    • You can zoom into a specific region of interest by clicking on the specific aberration that is shown within the Found Regions table. After this is done, a closer view of the aberration of interest is shown in the chromosome viewer.

      Alternately, use the zoom functions available in the toolbar or drag and stretch the red box on the ideogram to zoom in for a closer view of your data.
  • How did Illumina determine the chromosomal location for the potential/putative miRNAs?
    • To determine the chromosomal location for the potential/putative miRNAs, Illumina used the following procedure:

      1. Select 100% hits (of genome BLAST) for each mature miRNA sequence (if we don't get 100% match, a value of "zero" is shown in the bgx file)
      2. For each hit select the upstream and downstream pre-miRNA sequence (i.e., 5'-nnn-miRNA-nnnnnnnnnn-3' and 5'-nnnnnnnnnn-miRNA-nnn-3')
      3. Generate mfold structures for both upstream and downstream pre-miRNA sequences
      4. Analyze each structure (if the pre-miRNA will fold into the stem-loop structure)
      5. Filter out structures that are below the score threshold (estimated from Sanger training set)
      6. Select the structure with the best score for each blast hit site

      When multiple 100% hits are found in the genome for one particular mature miRNA sequence, the chromosomal coordinates are listed by the score, i.e., the location with the best score is listed first.

  • How do I access MiSeq Reporter?
    • MiSeq Reporter software runs as a web server that runs continuously on the MiSeq computer. To view current MiSeq Reporter status, open your web browser and enter the following URL: http://PCname:8042 (where “PC Name” is the name or IP address of the MiSeq PC).
  • How do I add more data (samples or lanes) to my GenomeStudio project?
    • Create a new GenomeStudio project with all of the samples and lanes you want to include.
  • How do I analyze TruSeq Synthetic Long-Read DNA data?
    • Purchase of the kit includes access to TruSeq Long-Read Assembly or TruSeq Phasing Analysis applications in BaseSpace.
  • How do I analyze data from a run on the MiSeq?
    • The system is designed to support multiple workflows inclusive of data analysis, which is performed on-instrument upon completion of the run. Output file formats are *.bcl, FASTQ, BAM, *.vcf, *.csv, and *.txt.

  • How do I analyze my Nextera Rapid Capture Enrichment data?
    • Nextera Rapid Capture Enrichment data can be analyzed using the HiSeq Analysis Software enrichment workflow for HiSeq data or MiSeq Reporter software for MiSeq data. See the HiSeq Analysis Software or MiSeq Reporter support pages for more information.

      Alternately, Illumina sequence base call output files (*.bcl) can be demultiplexed and converted to FASTQ format using the bcl2fastq converter software. The files can then be used for analysis with other third party software packages (e.g., BWA and GATK).

  • How do I analyze my TruSeq Targeted RNA Expression data?
    • Use MiSeq Reporter for on-instrument analysis. This software provides analysis of replicates and allows for pair-wise comparison of targeted regions.
  • How do I analyze my data for bisulfite sequencing applications?
    • Illumina suggests using a third-party methylation analysis solution such as Bismark, BSMap, or BS Seeker. See the Methylation Software section for more details.

  • How do I assess the quality of my RNA sample?
    • RNA quality is perhaps the most important factor in generating reliable and reproducible Real-Time PCR data. Traditionally, RNA quality was assessed using gel electrophoresis and comparing the 28S and 18S ribosomal RNA bands. Gel electrophoresis is a laborious, time consuming, and low-throughput method that requires fairly large amounts of RNA.

      Automated lab-on-chip capillary electrophoresis systems such as the Bioanalyzer (Agilent) and Experion (BioRad) have become popular tools for determining RNA quality. These systems use microfluidic technology to perform electrophoresis on glass chips at a miniaturized scale that overcome some of the issues of traditional electrophoresis. Data are presented as an electrophoretic trace of the RNA sample. The Agilent Bioanalyzer provides a quantitative measure of RNA integrity known as the RNA Integrity Number (RIN). A proprietary software algorithm examines the entire electrophoretic trace to determine RNA degradation and gives a numerical value between 1 and 10 to indicate RNA quality. An RNA sample with a RIN value of 10 is considered a highly intact sample where as a sample with a RIN value of 1 is considered a highly degraded sample.

  • How do I centrifuge the Eco plates?
    • The Eco sample loading dock comes with two plate adaptors. These plate adaptors have the same footprint as standard 96-well plates and can be used in standard centrifuge plate holders. Use both adapters to balance the centrifuge.

  • How do I change the instrument health upload option?
    • From the Welcome screen, select Menu, then Tools. The Options menu includes the checkbox to turn on or off instrument health data. Select View Terms for more information about the instrument health option.
  • How do I check the quality of my Nextera Rapid Capture Enrichment library?
    • Use an Agilent Technologies 2100 Bioanalyzer to check the quality and intended size distribution of a Nextera tagmented sample, the pre-enriched library, and the post-enriched library. For examples of bioanalyzer traces and library size distributions, see the Nextera Rapid Capture Enrichment Guide .
  • How do I check the quality of my library?
    • Illumina recommends the BioAnalyzer. Please see the Nextera DNA Sample Preparation Guide for more specific details and example traces.
  • How do I choose an appropriate reference gene?
    • Selection of a reference gene or genes is a critical step for expression analysis using Real-Time PCR. Validation of reference genes for each experimental condition is critical for obtaining accurate Real-Time PCR data. Validation requires determining if expression of the reference gene is stable between cells of different tissues and if any experimental treatment affects expression.

      The following paper contains a good summary of the validation process:

      Dheda K., et al. Validation of housekeeping genes for normalizing RNA expression in Real-Time PCR. Biotechniques 2004; 37: 112-119.

      The process starts with extracting and quantifying RNA samples from the samples under investigation (diseased vs. normal; treated vs. untreated). The next step is normalizing the input of RNA into the reverse transcription reaction. The expression of a panel of different reference genes is then measured by Real-Time PCR and the differences in Cq across the different samples is determined for each gene.

      There are a number of software programs available for selecting appropriate reference genes, including GeNorm (http://medgen.ugent.be/~jvdesomp/genorm/), BestKeeper (http://www.gene-quantification.de/BestKeeper-1.zip) and Norm-Finder (http://www.mdl.dk/Files/NormFinder_0953.xla).

  • How do I choose index combinations for my Nextera Rapid Capture Enrichment libraries if I only pool a small number of samples?
    • Please see the Low-Plex Pooling Guidelines section of the Nextera Rapid Capture Enrichment Guide for information on low-plex sample pooling.
  • How do I choose index combinations if I only pool a small number of samples with TruSeq sample prep?
    • Illumina uses a green laser to sequence G/T bases and a red laser to sequence A/C bases. At each cycle at least one of two nucleotides for each color channel need to be read to ensure proper registration. It is important to maintain color balance for each base of the index read being sequenced, otherwise index read sequencing could fail due to registration failure. For pooling strategies for a small number of samples, please refer to the "Adapter Tube Pooling Guidelines" section in sample prep guide for the kit you are using . Additionally it is recommended to create a sample sheet in the Illumina Experiment Manager (IEM) prior to performing sample prep in order to confirm appropriate index combinations not listed.

  • How do I contact Technical Support?
    • The fastest way to answer most questions is to refer to the Eco Support web page or in these FAQs. If you do not find an answer there, you can reach Technical Support by email at techsupport@illumina.com, by phone at 1.800.809.4566 in the U.S., or on the Illumina website at http://www.illumina.com/support.

  • How do I deselect MiSeq Reporter when I start a run?
  • How do I design primers/probes for a Real-Time PCR assay?
    • There are numerous primer design tools commercially available for purchase or freely accessible via the web. These tools simplify assay design significantly.

      Primers and probes for NuPCR, DBD, and HRM assays can be designed using Illumina's the web-based DesignStudio software (www.illumina.com/designstudio).

      Other primer design tools are Primer Express (Applied Biosystems), Beacon Designer (Premier Biosoft) and Real Time Design(BioSearch Technologies). Numerous websites contain databases of validated primer sets, including RTPrimerDB (http://medgen.ugent.be/rtprimerdb/) and the Quantitative PCR Primer Database (http://web.ncifcrf.gov/rtp/gel/primerdb/)

      If you design primers and probes manually, follow these criteria from the Primer Express manual:

      • The amplicon length should be between 50-150 bp to maximize amplification efficiency.
      • The optimal primer length is 20 nucleotides and the Tm should be between 58° and 60° C.
      • The optimal probe length is between 13 and 30 nucleotides for standard TaqMan probes and the Tm should be between 68° and 70°C.
      • Primer and probe sequences should not overlap.
      • The GC content of primers and probes should be between 30% and 80%.
      •  To prevent quenching, the 5' end of the probe cannot be a G residue.
      • Avoid runs of identical nucleotides. Primer and probe sequences must contain fewer than four consecutive G residues.
      • Ideally, to avoid genomic DNA amplification, primers should span an exon-exon junction in cDNA.
      Additionally, you should perform a BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi ) on primer and probe sequences to determine if the primers/probe will amplify sequences other than the target sequence.
  • How do I designate dual indices on a TruSeq sample sheet?
    • Designating dual indices on the sample sheet depends on whether a MiSeq or CASAVA sample sheet will be used. For MiSeq sample sheets, each index is entered into its own column. For CASAVA sample sheets, the indices are input in the format of "Index1-Index2". Please see the MiSeq Sample Sheet Quick Reference Guide or CASAVA User Guide for more information.

      Illumina recommends that you use the Illumina Experiment Manager (IEM) to generate your sample sheet. The appropriate index format is automatically entered based on the selected sample sheet type.

  • How do I determine if my RNA sample is contaminated with genomic DNA?
    • In Real-Time RT-qPCR, genomic DNA can potentially be co-amplified during the PCR reaction, contaminating the sample and leading to erroneous results. To determine if an RNA sample is contaminated with genomic DNA it is important to include a no-reverse transcriptase control during the RT step, and all RT-qPCR experiments should include a no-RT control. If the RNA sample is free of genomic DNA contamination the no-RT controls should not generate any signal after Real-Time PCR.

      To avoid genomic DNA contamination, treat RNA samples with DNAse before reverse transcription. Alternatively, design the PCR primers to anneal to sequences of the transcript that span a large intron. Primers designed in this way can only amplify cDNA.

  • How do I determine the efficiency of my Real-Time PCR assay?
    • The simplest and most commonly used method is the dilution or standard curve method. This method calculates PCR efficiency using the linear regression slope of a dilution series based on either of the following equations:

      E = 10(-1/slope) -1                               

      E = 10(-1/slope)

      The ideal slope is -3.32, which correlates to an amplification efficiency of 100%, meaning exactly one copy per cycle. Slopes in the range of -3.60 to -3.10 are generally considered acceptable for Real-Time PCR. These slope values correlate to amplification efficiencies between 90% (1.9) and 110% (2.1).
  • How do I determine which genes are accurately detected?
    • Filter the genes using the Detection p-value. Setting detection at .99 (p value <0.01) means that there is a 1% false positive rate.
  • How do I determine which version of genomeStudio software I am using?
    • With GenomeStudio software open, go to the Help menu and select About. The About screen includes GenomeStudio software version information.
  • How do I ensure I have an accurate amount of input DNA for the Nextera XT kit?
    • The Nextera XT DNA Sample Preparation Kit protocol is optimized for 1 ng of input DNA total. Illumina strongly recommends quantifying the starting genomic material. Nextera XT DNA Sample Preparation library preps use an enzymatic DNA fragmentation step and thus can be more sensitive to DNA input compared to mechanical fragmentation methods. The ultimate success of the assay strongly depends on using an accurately quantified amount of input DNA library. Therefore, the correct quantitation of the DNA library is essential.

      To obtain an accurate quantification of the DNA library, it is recommended to quantify the starting DNA library using a fluorometric based method specific for duplex DNA such as the Qubit dsDNA BR Assay system. Illumina recommends using 2 μl of each DNA sample with 198 μl of the Qubit working solution for sample quantification. Methods that measure total nucleic acid content (e.g. nanodrop or other UV absorbance methods) should be avoided because common contaminants such as ssDNA, RNA and oligos are not substrates for the Nextera XT assay.

  • How do I evaluate the success of a NuPCR reaction?
    • To determine PCR efficiencey and reproducibility, run a titration series of reactions using a positive control such as a plasmid containing the targeted sequence.
  • How do I filter and annotate my variants?
    • The Illumina VariantStudio filtering and annotation software is included with the purchase of TruSight DNA Amplicon Sequencing panels.
  • How do I find the largest aberration in my samples?
    • Click the Size column header to sort the data in the Found Regions table.
  • How do I find the region containing the greatest number of SNPs?
    • In the Found Regions table, click the # SNPs column header to sort the number of SNPs found in each region.
  • How do I find the smallest aberration in my samples?
    • In the Found Regions table, click the Clear filters button to allow aberrations as small as 1 kb to appear in your data. Then sort the data in the by clicking the Size column header.
  • How do I format my sample sheet?
    • All BaseSpace runs and any runs that require demultiplexing with either bcl2fastq or CASAVA require a CASAVA-formatted sample sheet. This format is described in the bcl2fastq User Guide and the CASAVA User Guide .

      Sample sheets for rapid runs include information for two lanes, as compared to eight lanes included in a sample sheet for a high output run. Sample sheets for rapid runs can be generated manually, using Excel or a text editor, or can be created by manually editing a CASAVA sample sheet generated with IEM.

      If you are using BaseSpace for data storage and analysis, a sample sheet is required for both rapid runs and high output runs. If using BaseSpace for run monitoring only, a sample sheet is not required.

      If you are analyzing data locally, rapid runs can be converted with bcl2fastq v1.8.4 without using a sample sheet, in which case the results are organized under a project directory named after the flow cell, and sample directories named after the lane numbers.
  • How do I get the Decode File Download Utility?
    • You can download the software free of charge from the Illumina iCom site. After logging into the system, click Downloads | Software. If you cannot access iCom or see the software in your list of downloads, please contact Customer Service for assistance.
  • How do I know if I have the most recent software versions? How do I get updates?
    • New software releases are announced via our IllumiNOTES e-newletter. You can request the latest versions by emailing the catalog number and your shipping and billing addresses to orders@illumina.com. Software updates are free for customers with a current service contract (warranty). Hot fixes are available for download from our website. You can also contact your Field Applications Scientist or technical support to find out the most recent software versions available.
  • How do I know if I need to throttle BaseSpace, and how do I apply throttling?
    • The BaseSpace Broker is designed to upload data to BaseSpace as soon as the data is generated on the HiSeq local drive. It will use as much bandwidth as is necessary to keep up with the data being produced. Under typical HiSeq run conditions, the upload of run data for storage and analysis will average less than 10Mbit/sec.

      In most cases, throttling of the BaseSpace Broker data upload is not necessary. Throttling can be necessary if greater control over network bandwidth usage is required, such as sites where instruments share the network with other users or sites with limited upload speed. Throttling might be necessary in certain scenarios where the local network connectivity is temporarily lost and then restored, which causes the BaseSpace Broker to suddenly consume more network bandwidth as it attempts to catch up with transfer of accumulated data. If no throttling is applied in such cases, the BaseSpace Broker might consume all available bandwidth on the network until the backlog of data is cleared. If throttling is applied and if the local network allows, Illumina recommends throttling to higher than the 10 Mbit/sec minimum specification. A recommended value of 20 Mbit/sec (approximately 3Mbytes/sec = 24Mbits/sec) allows the BaseSpace Broker enough bandwidth to recover, even if some delays in data transfer occur.

      If throttling is needed, provide the following instructions to your local IT administrator:

      Throttling of BaseSpace is performed on the HiSeq computer by application, rather than by IP address, as follows:

      1. In Windows, open a cmd window and open the Local Policy Editor. Run the program gpedit.msc.
      2. Expand the Computer Configuration / Windows Settings nodes.
      3. Select Policy-based QoS.
      4. Right-click Create new policy.
      5. Enter a name, such as Limit BaseSpace upload.
      6. Clear the Specify DSCP value.
      7. Select Specify Outbound Throttle Rate and enter 3 MBps (3 Mbytes/sec, or 24Mbit/sec), which is sufficient to allow data transfer to catch up.
      8. Click Next.
      9. Select Only applications with this executable name and enter Illumina.BaseSpace.Broker.exe.
      10. This policy applies to any source IP and target IP addresses. Click Next.
      11. This policy applies to all ports and protocols. Click Finish.
  • How do I know if I should be concerned about the Nextera Rapid Capture Custom Enrichment design in my targeted regions?
    • DesignStudio identifies Design Warnings to alert you to designs that are targeting areas of the genome that carry a higher performance risk. These warnings (e.g., low specificity, poor GC content) are displayed in the Regions, Targets, and Probes tables in DesignStudio.
  • How do I know if my network will allow me to upload data to the Illumina iControlDB?
    • We have created an application that we strongly recommend running before you attempt an upload. This will allow your network security settings to be examined, and for you to know if the security and firewalls will allow for uploading. This Upload Test App is available for download from the Illumina website.
  • How do I know if the TruSeq controls have worked?
      • The TruSeq controls were not designed to be a qualitative metric of the efficiency of the various steps in the sample prep but rather an indicator of whether or not the step worked or not. Actual counts of the controls will vary based on sample type, input, etc.
      • In SAV (Sequencing Analysis Viewer), if you see counts for a particular control, this mean that this step has worked whereas if you see no counts (dark blue color), this step has likely failed. If the band has been cut across multiple insert sizes, you may see counts at different size increments.
  • How do I know if the quality of my DNA is sufficient for Illumina microarray assays?
    • For the Infinium Assay, we recommend fragment sizes of at least 2 kb. The GoldenGate Assay can tolerate shorter fragments (> 200 bp) and is more forgiving of degraded samples.
  • How do I know that MiSeq Reporter has completed its analysis?
    • When MiSeq Reporter analysis is finished, a checkmark appears in the State column of the Analyses tab and the CompletedJobInfo.xml file is written to the root level of the analysis folder.
  • How do I make sure that my HiSeq is ready to send data to BaseSpace?
    • To upload data to BaseSpace from a HiSeq, a minimum upstream connection of 10 Mbit/second per instrument is needed. Network speed can be assessed by using free online tools such as www.speedtest.net.
  • How do I merge data from two flow cells?
    • Using CASAVA: To merge data from different flow cells (different runs), use the configureBuild script in CASAVA v1.8.2. First, align the data (samples) from each flow cell separately using configureAlignment. Then, include each sample directory as an input directory in the configureBuild.pl command line. Input directories are specified by the –id option, as detailed on page 100 of the CASAVA v1.8.2 User Guide (Rev C).

      Using BaseSpace: BaseSpace includes a Sample Merge function that allows you to merge data from a single sample originating from different flow cells. This merging is performed before alignment analysis of the sample data.
  • How do I normalize between samples for TruSeq Targeted RNA Expression?
    • In order to normalize your data, identify a gene from RNA-Seq or Microarray data that is constant across the samples being tested. Include assays that target this gene in your TOP panel when using DesignStudio. Additionally, this gene needs to be identified in the sample sheet, in a column with the header “Normalize”, so MiSeq Reporter will use it to normalize the data.
  • How do I obtain genotypes from my data with KaryoStudio?
    • Genotype information is not provided by KaryoStudio. To obtain the SNP genotypes from your data, you must load your data into the GenomeStudio Genotyping Module. For more information, refer to the GenomeStudio Genotyping Module V1 .0 User Guide .
  • How do I order probes for TruSeq Targeted RNA Expression?
    • Use DesignStudio to order probes. You will need a MyIllumina account.
  • How do I perform TruSeq RNA without using PCR to enrich DNA fragments that have adapter molecules on both ends?
    • The number of PCR cycles should be minimized to avoid skewing the representation of the library.  Illumina recommends 15 cycles of PCR, which has been shown to provide the best performance in terms of coverage, reproducibility, and quantity of material.  Although not recommended by Illumina, if you want to eliminate PCR, begin with a large amount of starting material (~5 μg) to generate equivalent yield as the standard protocol.  Note that at least one cycle of PCR is required to open the forked adapters. 

  • How do I perform amplicon sequencing on a MiSeq?
    • The TruSeq Custom Amplicon Kit uses a highly multiplexed assay to generate up to 384 amplicons across many samples with integrated barcodes for pooling prior to sequencing on a MiSeq. The Nextera XT DNA Sample Prep Kit can be used to prepare user-generated amplicons with standard PCR.

  • How do I place an order for a custom OPA?
  • How do I prevent the magnetic beads from drying out during GoldenGate processing?
    • To avoid this, decrease the number of columns processed at one time during each of the wash and buffer exchange steps. To resuspend beads that have partially dried, re-vortex the ASE plate containing the UB2 buffer in the Add Mel step and/or the IP1 during the Inoc PCR step.
  • How do I quantify TruSeq DNA PCR-Free libraries?
    • The TruSeq DNA PCR-Free protocol does not contain a PCR enrichment step to select for completed ligation products, therefore quantitation must be done by qPCR as other methods will quantify fragments that may not have adapters at both ends and will thus not generate clusters or sequence. See the Consumables and Equipment section of the TruSeq DNA PCR-Free Sample Preparation Guide for the required KAPA Library Quantification Kit. Follow qPCR instructions included in the KAPA Library Quantification Kits for Illumina sequencing platforms Technical Data Sheet using the KAPA standard, with the modifications specified in the Validate Library sections of the TruSeq DNA PCR-Free Sample Preparation Guide .
  • How do I quantify my RNA sample?
    • The most widely used method to quantify RNA is traditional UV spectroscopy. A diluted RNA sample is quantified by measuring its absorbance at 260 nm and 280 nm. The concentration is calculated using the equation:

                                                      [RNA] μg/ml = A260 x dilution factor x 40

                                              where 40 is the average extinction coefficient for RNA

      In addition, the A260/A280 ratio can be used to estimate RNA purity. An A260/A280 ratio between 1.8 and 2.1 indicates a highly pure RNA sample.

      UV spectroscopy is relatively simple to perform but has several drawbacks. It does not discriminate between RNA and DNA so it is advisable to DNAse treat RNA samples before quantifying. DNA in the sample will lead to an overestimation of RNA concentration. Since proteins and residual phenol from the purification can interfere with absorbance readings, it is important to remove these contaminants in purification. Also, absorbance readings are dependent on pH and ionic strength. Dilute RNA samples in TE (pH 8.0) and use TE to blank the spectrophotometer before taking absorbance readings.

      An alternative method for quantifying RNA samples is to use fluorescent dyes such as RiboGreen (Invitrogen). RiboGreen exhibits a strong fluorescent signal when bound to nucleic acids. Samples are quantified in a fluorescence microplate reader or standard spectrophotometer relative to a nucleic acid standard curve of known concentration. The linear range of quantification using RiboGreen is three orders of magnitude, from 1 μg/ml down to 1 ng/ml. The major advantage of fluorescent dyes over absorbence-based methods is that it is not affected by contaminating proteins or organic solvents carried over from the purification process. DNAse treatment is still recommended as RiboGreen does not discriminate between RNA and DNA.

  • How do I quantify the final TruSeq Nano DNA libraries?
    • qPCR or fluorometric quantification assays using dsDNA binding dyes (such as Qubit or Picogreen) can be used as methods of final library quantification.
  • How do I read the TruSeq Sample Prep controls?
    • RTA 1.9 (on GA) or 1.10 (on HiSeq) or higher processes the controls. To visualize them, SAV (Sequencing Analysis Viewer) 1.7 or higher must be used. RTA produces a new InterOp metrics file called ControlMetrics; this is the file SAV uses to get control counts.

  • How do I requeue a run for analysis in MiSeq Reporter?
    • MiSeq Reporter needs to have access to the repository, which is the location of the folder containing data for analysis. You can set this location in the settings window on the MiSeq Reporter main screen. When MiSeq Reporter has access to the repository, your runs appear in the Analyses tab in MiSeq Reporter. Select the Requeue checkbox next to the run you want to analyze, and then click Requeue to start analysis.

  • How do I select the right number of index cycles and chemistry for sequencing dual-indexed libraries in HCS 1.5?
    • In order to perform dual-index sequencing in HCS 1.5, select the TruSeq Dual Index Sequencing Primer Box from the Index chemistry drop down menu on the recipe screen. This selection enables the use of the required chemistry for sequencing dual-indexed libraries, and must be used for sequencing any dual-indexed libraries (Nextera or TruSeq HT) regardless of which sequencing primers you will use for your run. Selecting any other setting will result in less than an eight-cycle index read.

  • How do I send run data to BaseSpace?
    • Run data can only be uploaded to BaseSpace if the BaseSpace option is selected during run setup in the HiSeq Control Software. See the HiSeq 2500 System User Guide (part # 15035786) for information on setting up a run with a connection to BaseSpace.

      For more information on BaseSpace, or to set up a free BaseSpace account, see https://basespace.illumina.com/home/index.

  • How do I set up runs so that secondary analysis is not performed on instrument but is performed solely by MiSeq Reporter running on another computer?
    • To skip analysis (everything after FASTQ generation), you can specify the GenerateFASTQ workflow in the sample sheet. On the external computer, set the repository location using the Settings window on the MiSeq Reporter interface or edit MiSeq Reporter.exe.config file. The repository is the folder containing data files ready for analysis.
  • How do I set up standard concentrations with non-serial dilutions?
    • There are two methods for setting up non-serial dilutions or dilutions with a factor greater than 10. The following list shows a set of non-serial dilutions:

      • Standard 1 with concentration as 44500000
      • Standard 2 with concentration as 4450000 (1:10 dilution)
      • Standard 3 with concentration as 2225000 (1/20 dilution)
      • Standard 4 with concentration as 1112500 (1/40 dilution)

      Automated method: Click the Standards button beside your standard assay to open the Set Up Standards pane, and then click Define Standards to open the Dilutions dialog box. Enter "4" for the number of points, 4450000 as the starting quantity, and 10 as the dilution factor. Back in the main Set Up Standards pane, directly type the correct values for the last two dilutions into the appropriate fields.

      Manual method: Click the Standards button beside your standard assay to open the Set Up Standards pane. Type 4450000 directly into the first field, and then press Enter to make the next field active. Type the second quantity into that field and continue until you have entered all four quantities.

      The following list shows a set of serial dilutions:

      • Standard 1 with concentration as 10000
      • Standard 2 with concentration as 1000
      • Standard 3 with concentration as 100
      • Standard 4 with concentration as 1

      To enter serial dilutions with a dilution factor between 2 and 10, click the Standards button beside your standard assay to open the Set Up Standards pane. Click Define Standards to set up the serial dilutions.

  • How do I set up the sample sheet for MSR analysis?
    • Use the Enrichment workflow to create a sample sheet formatted for analysis of TruSight One data. Refer to the IEM TruSight One or TruSight Rapid Capture Quick Reference Card for instructions on creating your sample sheet.

  • How do I transfer my DesignStudio projects for TruSeq Custom Enrichment or Nextera Custom Enrichment for use in the DesignStudio module for Nextera Rapid Capture Custom Enrichment?
    • For transfer instructions, see the DesignStudio Update for Nextera Rapid Capture Custom Enrichment .
  • How do I use BaseSpace for run monitoring only, such as SAV functionality in BaseSpace?
    • Run monitoring with BaseSpace is selected during run setup.
  • How do I use BaseSpace for run monitoring?
    • The Run Monitoring BaseSpace option allows you to remotely monitor a run in progress by logging in to your BaseSpace account. You need to select the Run Monitoring option during run setup. Then, log in to your BaseSpace account from anywhere and view your run in the BaseSpace version of Sequence Analysis Viewer (SAV).
  • How do I use the Decode File Download Utility to download files for specific chips that are already in my lab without seeing such a long list?
    • Click BeadChips by Barcode on the Main tab to open a text box. You may use the barcode scanner from any Illumina workstation to barcode-scan the chips into the system, or simply type them into the box. This feature only retrieves the files for the barcodes you specify.
  • How do I view genoplots with SNP information?
    • You must load your data into the GenomeStudio Genotyping Module.
  • How do I visualize my Nextera Rapid Capture Enrichment designed targets in a genome browser?
    • Use the DesignStudio table Export function to save designed regions, targets, and probes in .bed file format. Exported BED files can be imported into a genome browser, such as the Broad Institute Integrative Genome Viewer (IGV), which can be downloaded and run on your local PC computer, or the online UCSC Genome Browser.

      Note:

      • The UCSC Genome Browser may not accept very large BED files with many annotations.
      • Illumina recommends adding the track name annotation to your BED files when viewing multiple custom tracks, such as probes and gaps, in the UCSC Genome Browser. BED files exported from DesignStudio do not contain a header with a defined track name. Therefore, when loaded into the UCSC Genome Browser, they appear with the generic track name of 'User Track' and description of 'User Supplied Track' and will overwrite any other previously uploaded unnamed tracks.
  • How do I visualize my TruSight One targets in a genome browser?
    • TruSight One regions, targets, and probes are provided in *.bed file format and can be found by visiting the TruSight One Product Support page.  The *.bed file can be imported into either the UCSC Genome Browser (genome.ucsc.edu) or the Integrated Genome Viewer (IGV) (www.broadinstitute.org/igv/).

  • How do Nextera DNA Sample Preparation Kits work?
    • Nextera sample preparation kits are used to prepare DNA samples for next‐generation sequencing. These kits use in vitro transposition to prepare sequencer‐ready libraries from genomic DNA for all Illumina sequencing platforms. The technology simultaneously fragments and tags DNA in a single tube reaction and this process is referred to as "tagmentation".  The protocol takes approximately two hours for a 24 sample prep, with ~60 minutes of hands-on time, and requires 50 ng of starting DNA.

  • How do data compression options in HCS v2.2 change data analysis or data handling?
    • Because run output has zipped BCL files, you have to use the bcl2fastq v1.8.4 conversion software to perform BCL to FASTQ conversion on your local Linux analysis system. This tool is run on Linux and has the same syntax, options, and functions (including demultiplexing) as the configureBclToFastq.pl script of CASAVA. The only difference is that it can be used to analyze either zipped or non-zipped BCL files.

      If you send your data to BaseSpace, BCL to FASTQ conversion and demultiplexing are performed automatically following the completion of the data upload.
  • How do the TruSeq Stranded mRNA and Total RNA protocols generate stranded cDNA?
    • Strand specificity is achieved by replacing dTTP with dUTP in the Second Strand Marking Mix (SMM). The incorporation of dUTP in second strand synthesis effectively quenches the second strand during amplification, since the polymerase used in the assay will not incorporate past this nucleotide. Further specificity is achieved by addition of Actinomycin D to the First Strand Master Mix Act D (FSA). Actinomycin prevents spurious DNA dependent synthesis during first strand synthesis, while allowing RNA dependent synthesis.
  • How do you achieve multiplexing with the VeraCode Beads?
    • Multiplexing is achieved by pooling together beads with unique codes. Illumina offers standard products to enable users to develop anything from a single-plex to several hundred-plex reactions per sample in a single well.
  • How do you ensure that different adapters are ligated to each end of a DNA fragment using the TruSeq DNA PCR-Free protocol?
    • Illumina's proprietary method ensures ligation of two different adapters in the required orientation to opposing ends of a DNA fragment. Fragments without the proper adapters ligated at both ends may remain due to the absence of PCR enrichment that selects for completed products. Ligation is ensured by performing a qPCR step at the end of the protocol. qPCR will only quantify libraries that have complete adapter ligations and will cluster and sequence. Other methods will quantify fragments that may not have adapters at both ends and will thus not generate clusters or sequence. Quantification by methods other than qPCR will be inaccurate.
  • How do you ensure that different adapters are ligated to each end of a DNA fragment using this protocol?
    • Illumina’s proprietary method ensures ligation of two different adapters in the required orientation to opposing ends of a DNA fragment. PCR selects for these and finalizes the construct ready for hybridizing onto the flow cell surface. Adapter sequences can be determined by sequencing the ligation fragments, but sequence information alone is not sufficient to uncover the method.
  • How do you ensure you have enough markers for linkage mapping?
    • This is an optimal set of markers for linkage mapping. By simulation studies, it has been suggested that a 1 to 2 cM bi-allelic map of polymorphic markers (minor allele frequency 20–50%) will extract most of the inheritance information and that for common linkage study designs, adding more markers provides diminishing returns (Kruglyak, 1999). In a study of 188 meioses, the average information content over all chromosomes was over 97% and never dropped below 83%. This high information content throughout the genome can be attributed to both the appropriate level of marker density and high heterozygosity of SNPs used in the panel and will ensure maximal power for detecting linkage to a disease or trait and defining the linkage interval.
  • How does DesignStudio select probes?
    • For TruSeq Custom Amplicon, optimal probes are chosen using an algorithm that considers melting temperature (Tm), % GC, length, secondary structure, uniqueness in the genome, and the presence of underlying SNPs (based on dbSNP). For additional information, refer to the online help functionality in DesignStudio.
  • How does Illumina define a validated assay?
    • Assays marked validated in DesignStudio have been shown to give results consistent with fold changes observed by RNA-Seq in testing across human tissues.
  • How does Illumina select SNPs and develop the SNP genotyping assays?
    • For custom genotyping projects, we will validate and develop the SNP genotyping assays for you. Prior to development, we will work with you to select SNPs, screening those SNPs informatically. Assay development success depends upon the source of the SNP, its frequency in the population, and the assay system. For instance, many SNPs derived from databases are sequencing errors, or exist in too low a frequency to be useful in most genotyping studies.
  • How does Isaac improve the speed of analysis 4–6 times over existing methods?
    • The Isaac aligner aligns reads by first identifying a small but complete set of relevant candidate mapping positions. The Isaac aligner begins with a seed-based search, using 32-mers as seeds. The initial single-seed search is followed by a multi-seed only for the reads that couldn't be placed unambiguously with a single seed. Speed up is achieved by sorting the reference index by the 32-mers.  Improvement to accuracy is achieved by flagging of all the ambiguous reference positions in the index.

      Following the seed-based search, selection of the best mapping among all the candidates is performed. For paired-end data sets, all mappings where only one end is aligned  (orphan mappings) trigger a local search to find additional mapping candidates (shadow mappings) in the neighborhood defined by the expected minimum and maximum insert size.  After optional trimming of low quality 3' ends and adaptor sequences, the possible mapping positions of each fragments are compared, taking into account pair-end information when available, possible gaps (using a banded Smith-Waterman gap aligner) and possible shadows. The selection is based on the Smith-Waterman score (using BWA, ELAND or user-defined scores) and on the log-probability of each mapping. The main speed-up comes from a parallel implementation of the gap aligner (using the SSE2 instruction set) and a shadow aligner optimized for short inserts. Further improvements could be achieved with AVX. The gapped alignment could be delegated to a coprocessor (e.g. Xeon Phi or GPU), however it is unclear if the benefit of large-scale parallelization would outweigh the cost of transferring the data between host and coprocessor.

      Following alignment the fragments are sorted. Major speed-up in the sorting speed-up comes from efficient binning of the selected mappings, which greatly simplifies the sorting.  Further analysis is performed to identify duplicates and optionally to re-align indels.

  • How does KaryoStudio determine which aberrations go into a report?
    • After you set how many aberrations should go into the report, KaryoStudio shows the aberrations based upon descending size. Only found regions with a checkmark in the Found Regions Table are displayed in the report.
  • How does TruSeq RNA Access Library Prep handle ribosomal RNA contamination?
    • Because it enriches the coding transcriptome, the ribosomal RNA is washed away during the wash procedures.
  • How does TruSeq Synthetic Long-Read DNA library prep technology work?
    • The TruSeq Synthetic Long-Read DNA Library Prep and Barcode kits are designed for two applications; preparing DNA libraries for de novo synthetic long-read sequencing and analysis and phasing analysis from whole human genome sequencing data. The kits combine TruSeq and Nextera chemistries to prepare DNA libraries for sequencing and informatics apps to aid in performing long reads assembly or phasing analysis.
  • How does VariantStudio handle tri-allelic sites?
    • When both alleles of a heterozygous position are different from the reference, as in a tri-allelic position, the variants are split into two lines and both variants are annotated.

  • How does changing Cluster Distance in the Clustering Options dialog box affect my results?
    • Cluster distance specifies the maximum distance that samples can be away from each other and still considered part of the same cluster. Increasing cluster distance will result in fewer clusters that are larger in size, while decreasing cluster distance will result in more clusters which are smaller in size. A cluster distance of 0.06 is typically a good starting point for initial clustering.

  • How does dual indexing work?
    • For paired-end flow cells, dual indexing now requires 23 additional cycles of sequencing - eight cycles for the Index 1 (i7) Read, eight cycles for the Index 2 (i5) Read, plus seven non-imaging, chemistry-only cycles at the beginning of the Index 2 (i5) Read. For single-read flow cells, dual indexing only requires 16 additional cycles of sequencing - eight cycles for the Index 1 (i7) Read and eight cycles for the Index 2 (i5) Read.
  • How does oligo design work for TruSeq Targeted RNA Expression? Can I design custom oligos to any gene of my choosing?
    • The TruSeq Targeted RNA Expression assay consists of predesigned probes. Once a gene is selected, DesignStudio provides a list of predesigned probes. Due to the predesigned nature of these probes, DesignStudio design time is negligible. Options for probes include:
      • Transcript specific
      • 5' end
      • 3' end
      • cSNP
  • How does the Eco evaluation plate work?
    • The Eco evaluation plate contains PCR primers that are designed to detect and quantify an artificial DNA sequence, with template DNA at defined quantities or no template at all. A standard curve with 20000, 10000, 5000, 2500, and 1250 copies in quadruplicate is used to quantify an unknown population of 24 replicates.

      A user simply needs to add 20 µl of master mix to each well at a 1X concentration, incubate to fully resuspend the lyophilized primers and template, then centrifuge the plate and load it into the Eco. The Eco software comes with a preloaded template run file that includes the plate layout as well as the thermal cycling conditions.

      Upon analysis, the data will show PCR efficiency, R2, standard deviation of replicates, and the melt curve analysis. The plate is intended for demonstration purposes, software training, and validation.

  • How does the Eco system achieve higher HRM performance than other block-based systems?
    • Temperature control and uniformity are the most challenging factors affecting HRM. The Tm shift for a single base change can be as small as 0.2° C for challenging Class IV A to T single nucleotide polymorphisms. Most current block-based instruments report temperature uniformity specifications in the range of ± 0.25° C to ± 0.5° C, generally considered too high to reliably differentiate a Class IV SNP. Block-based instruments that claim to perform HRM do so by extensive calibration and software compensation or by employing specialized analysis methods, such as temperature shifting, to overcome the thermal non-uniformity across the block.

      The Eco system's unique thermal block provides thermal uniformity of ± 0.1° C, well above the industry standard for a block-based system. This extreme thermal uniformity allows the Eco system to perform HRM without software corrections. The thermal uniformity of the Eco system supports genotyping of even the most challenging Class IV SNPs.

  • How does the Eco system provide a significantly higher level of thermal uniformity compared to other block-based systems?
    • Eco's hollow thermal block is constructed of highly conductive silver and is filled with a thermal conductive fluid. The fluid is rapidly circulated through the block by paired agitators that are powered by high-efficiency electromagnetic motors. The constant circulation of fluid evenly distributes the heat throughout the block to remove the hot or cold spots typical of block-based instruments. This design allows for rapid temperature ramps and extreme thermal uniformity (± 0.1° C) well to well across the plate, with no edge effect.

  • How does the Isaac human WGS workflow compare to BWA / GATK in terms of sensitivity and specificity?
  • How does the Ribo-Zero portion of the TruSeq Stranded Total RNA Sample Prep protocol work?
    • TruSeq Stranded Total RNA Sample Prep Ribo-Zero kits remove ribosomal RNA (rRNA) using a hybridization/bead capture procedure that selectively binds rRNA species using biotinylated capture probes. The probe:rRNA hybrid is then captured by magnetic beads and removed using a magnet, leaving the desired rRNA-depleted RNA in solution.

  • How does the assay work?
    • One pair of oligos is designed for each amplicon. Hybridization of these oligos to unfragmented genomic DNA occurs in a 96‐well plate, followed by extension and ligation to form DNA templates consisting of the regions of interest flanked by universal primer sequences. Using indexed primers supplied with the kit, DNA templates are then PCR amplified, pooled into a single tube, and sequenced on the MiSeq System. For additional details on the assay, please refer to the User Guide.

  • How frequently do you update your annotation database?
    • Illumina does not have a specific schedule for updating the annotation database. This process largely depends on the cadence of updates from the different sources aggregated to create the annotation database.

  • How is Target Region Coverage calculated in Design Studio?
    • It is calculated as the proportion of bases in the requested Target Region that will be sequenced by the designed amplicon. The region sequenced by the designed amplicon includes the nonvariable flanking probes and sequence in between.
  • How is base calling and quality scoring performed on a NextSeq 500?
    • Primary analysis is performed by a new implementation of real-time analysis (RTA), RTA 2.0, which includes important architecture differences from RTA on other Illumina sequencers. For example, all processes are performed in memory to maximize processing speed, and configuration files and output file formats are different. For more information, see the NextSeq 500 System User Guide.

      The primary analysis workflow includes the following steps:

      • Template generation—Defines the position of each cluster on a flow cell tile. A tile is one field of view of the camera.
      • Imaging—In real time, images are passed in memory to RTA 2.0 as tiles.
      • Base calling—Determines a base for every cluster of a given tile at a specific cycle. After a base is called, RTA 2.0 assigns a quality score. The result is written to base call (BCL) files, which are aggregated in one file per lane per cycle.
  • How is flow cell clustering conducted on MiSeq?
    • Cluster generation follows the same process that occurs on the cBot in preparation for runs on the HiSeq or Genome Analyzer, except on MiSeq all reagents for cluster generation, sequencing, and paired-end chemistry are loaded onto the instrument in a pre-filled reagent cartridge prior to starting the run. When the run is started, the MiSeq performs cluster generation followed by sequencing and paired end chemistry (if applicable).

  • How is specificity achieved for mature miRNA over pre-miRNA?
    • Specificity is achieved in the Extension step (in the Add MEL step) and cycling. The Extension step favors mature miRNAs because longer sequences will not achieve complete extension to the PCR primer portion of the Make CSP poly-dT primer to the same degree as mature miRNAs.
  • How is strandedness maintained after DNA amplification?
    • Strandedness is maintained via the directionality of the adapters. The p7 adapter will be on the 3' end of the cDNA strand. As a consequence, the cDNA strand is sequenced. Second strand synthesis is performed using dUTP in place of dUTP and a high fidelity polymerase that cannot process through dUTP template is used for PCR enrichment. As a result, only the first strand product is amplified and the strand information is retained based on the p5 and p7 adapter orientation.
  • How is the DNA fragmented?
    • Nebulization is a very reproducible process that produces random fragmentation. Alternative fragmentation methods may be more appropriate depending on sample availability. Other available technologies include sonication, ultrasound, and hydroshearing.
  • How is the Illumina Nextera kit different from the legacy Nextera kit from Epicentre?
    • The Illumina Nextera kit has improved master mixes resulting in an easier protocol with less tubes. Additionally, there is an optimized PCR that does not require the purchase of any additional components and an optimized tagmentation reaction. Finally, the new dual indexing strategy allows 96 indices per lane and samples can be sequenced on all Illumina sequencing platforms.
  • How is the TruSeq ChIP Sample Prep kit different from the legacy ChIP-Seq Sample Prep kit?
    • The TruSeq ChIP Sample Prep kit generates Paired End (PE), indexed libraries that are compatible with all Illumina sequencing platforms and can be multiplexed. The legacy ChIP-Seq Sample Prep kit generates Single Read (SR) and non-indexed libraries that are compatible with Genome Analyzer, HiSeq and HiScanSQ, but not MiSeq.

  • How is the TruSeq RNA Access Library Kit different from the TruSeq Stranded Total RNA and TruSeq Stranded mRNA sample prep kits?
      • TruSeq RNA Access Library Prep is similar to TruSeq Stranded Total RNA Sample Prep, because it enables RNA sequencing from low quality and FFPE-derived samples, and maintains strand information. Unlike TruSeq Stranded Total RNA Sample Prep, TruSeq RNA Access Library Prep captures only the coding regions of the transcriptome, allowing much higher throughput and requiring lower sequencing depth.
      • TruSeq RNA Access Library Prep is similar to TruSeq Stranded mRNA Sample Prep, because  it offers sequencing of the coding RNA and does not require ribosomal depletion.
      • TruSeq RNA Access library prep is compatible with low quality and FFPE-derived samples because it does not rely on the presence of a poly-A tail.
  • How is the beta value calculated on the Infinium HumanMethylation450 BeadChip?
    • The beta value (β) is used to estimate the methylation level of the CpG locus using the ratio of intensities between methylated and unmethylated alleles.

  • How is the instrument health upload option communicated to customers?
    • The first time the HCS 2.2 is launched, you will see a notification regarding instrument health data. This notification will appear only once during the first initialization of the HCS and will not appear again. Note that in pre-release, early access versions of HCS 2.0, this notification does not appear. If the customer is not present to view this notification, Illumina staff will refer the customer to the Options menu and ensure that they are aware of this setting.
  • How is the study number determined for the samples I have uploaded?
    • The study number assigned to your samples is the next study number available in sequence.

  • How large is the gap size between the Upstream Locus Specific-Oligo and the Downstream Locus Specific-Oligo for TruSeq Targeted RNA Expression?
    • On average, the gap size is 15 bases, but can be as small as 1 and as large as 43 bases. The average insert size is 67 bp. The average length of the ULSO and DLSO are 25 bases, not including the PCR primer binding sites.
  • How large is the run folder?
    • Saving the standard set of files without thumbnails results in a run folder that is approximately 200–500 GB. If standard thumbnails are saved, approximately an additional 300 GB is required. Approximately 1 TB is storage used by the alignment folder and related folders.
  • How long are decode files available via the Decode File Download Utility?
    • Files appear on the server approximately 24 hours after they are shipped. We guarantee that files remain on the server for at least 30 days from the time they are shipped. Downloading the files changes their status in the database so you can filter them, but you can download files as many times as you like as long as they remain on the server.
  • How long are the upstream and downstream probe sequences?
    • The length of the upstream and downstream probe design can vary between 22 bp and 30 bp, regardless of what size amplicons are being designed.
  • How long can HiSeq v4 reagents be stored after initial preparation?
    • Prepare HiSeq v4 SBS reagents the night before or on the same day of use only. Do not store reagents longer than overnight. For use on the same day, store prepared reagents on ice in the original bottle with the cap tightened. For use the next day, store prepared reagents at 2°C to 8°C overnight.

  • How long can a Rapid flow cell be stored after finishing template hybridization and first extension on the cBot and before being run on the HiSeq 2500?
    • You can store the Rapid flow cell up to 24 hours after template hybridization and first extension. However, Illumina recommends that you start the sequencing run on the same day.
  • How long can rapid run SBS reagents be stored after initial preparation?
    • Prepare rapid run SBS reagents the night before or on the same day of use only. Do not store reagents longer than overnight. For use on the same day, store prepared reagents on ice in the original bottle with the cap tightened. For use the next day, store prepared reagents at 2°C to 8°C overnight.
  • How long do you expect the Eco system LED array to last? Can Illumina provide a lifetime warranty for the LED array?
    • The LED arrays are designed to outlast the life of the instrument and are covered by the instrument warranty.

  • How long does it take for MiSeq secondary analysis to complete?
    • For a 2 x 250 bp run, analysis takes about 3 hours. This timing is dependent on running the latest PC RAM configuration on the MiSeq. This also depends on the genome size for resequencing. If analysis is taking longer than two hours, consider mapping to a more appropriate reference for your sample, or perform analysis offline by installing MiSeq Reporter on another computer.

      If an alignment is performed against the whole genome, then the analysis time will be significantly longer than two hours. Also, bioinformatics analysis for metagenomics may take as long as 12 hours.

  • How long does it take from the start of the run until I have cluster density metrics?
    • Depending on cluster density, metrics appear at the beginning of cycle 20, if you are running MCS v2.3. With earlier version of MCS, metrics appear at the beginning of cycle 7.
  • How long does it take to complete a 150-cycle run on the MiSeq?
    • A 150-cycle run takes approximately 20 hours.

  • How long does it take to complete a 600-cycle run on the MiSeq?
    • A 600-cycle run takes approximately 55 hours.

  • How long does it take to complete a run on the MiSeq?
  • How long does it take to complete a run on the NextSeq 500?
    • Run duration depends on the number of cycles sequenced, the type of flow cell, and whether the sequence is single or paired end. For example, a 2 x 150 bp run on a high-output flow cell takes approximately 29 hours, while a 1 x 75 bp run takes less than 11 hours. For more information, see the NextSeq 500 specifications page.

  • How long does it take to complete a water wash and maintenance wash?
    • Wash protocols have not changed between TruSeq v3 and HiSeq v4 configurations. A water wash take approximately 1 hour, and a maintenance wash take approximately 3 hours.
  • How long does it take to generate clusters on a flow cell?
    • It takes approximately four hours on cBot and about five hours on the Cluster Station.
  • How long does it take to generate ready-to-sequence Nextera Rapid Capture Enrichment libraries?
    • It takes 1.5-2 days (approximately 30 hours) from genomic DNA input until libraries are ready to load on the flow cell, including approximately 5 hours of hands on time. Nextera Rapid Capture Enrichment libraries can be loaded on the flow cell the afternoon of day 2 of the protocol.

  • How long does it take to generate ready-to-sequence TruSeq RNA Access libraries?
    • It takes 2 days for 8-16 samples and 3 days for 17-48 samples, from total RNA input until libraries are ready to load on the flow cell. This includes approximately 11 hours of hands-on time.
  • How long does it take to perform a post-run wash?
    • The post-run wash takes approximately 30 minutes.
  • How long does it take to perform cluster generation and primer hybridization on the MiSeq?
    • This step takes approximately 70 minutes.
  • How long does it take to perform one full cycle on the MiSeq?
    • One full cycle takes approximately 6 minutes and consists of a chemistry cycle and an imaging cycle.
  • How long does it take to ship Nextera Rapid Capture Custom Enrichment reagents after placing the order in DesignStudio?
    • Typically, it takes 3–4 weeks to manufacture and ship a Nextera Rapid Capture Custom Enrichment Kit order.
  • How long does it take to switch between modes on my HiSeq system?
    • It takes about 45 minutes to change from a high output (TruSeq v3 or HiSeq v4) to TruSeq Rapid mode. It takes about 4 hours to change from TruSeq Rapid to a high output (TruSeq v3 or HiSeq v4). Time spent for mode switching is in addition to instrument washing performed at the end of each run.
  • How long does template generation take on the MiSeq?
    • With MCS v2.3, template generation occurs during the first 7 cycles of sequencing. When using earlier versions of MCS, template generation occurs during the first 4 cycles of imaging.
  • How long does the Nextera Enrichment sample prep take?
    • Samples can be prepared in less than 3 hours and complete enrichment of up to 96 samples can be completed in 2.5 days.
  • How long does the TruSight DNA Amplicon assay take?
    • The assay takes less than eight hours total, with 2–3 hours of hands on time. TruSight DNA Amplicon libraries are ready to run on an Illumina sequencer without any further manipulation.
  • How long does the assay take?
    • The assay takes less than 8 hours total, with 2–3 hours of hands-on time. TruSeq Custom Amplicon libraries are then immediately compatible with the MiSeq system without any further manipulation.
  • How long does the cBot take to perform clustering on a HiSeq v4 flow cell?
    • Clustering takes slightly more than 2 hours. Clustering on a HiSeq v4 requires the updated cBot software (v2.0.16, or later) and requires v9.0 recipes.
  • How long does the cBot take to perform the template hybridization procedure on a 2 lane flow cell?
    • Just under one hour.
  • How long does the cluster generation step take on the NextSeq 500?
    • Cluster generation is the first step in the sequencing run in which single DNA molecules are bound to the surface of the flow cell and then bridge-amplified to form clusters. Cluster generation on the NextSeq 500 takes about two hours and twenty minutes.
  • How long does the resynthesis step take on a HiSeq?
    • The resynthesis step takes approximately 3 hours.
  • How long is each Index Read?
    • Both Index 1 (i7) and Index 2 (i5) are 8 bp in length.
  • How long is each index read for TruSeq HT dual-indexed libraries?
    • Both Index 1 (i7) and Index 2 (i5) are each 8 bp in length.

  • How long will it take to download the files using the Decode File Download Utility?
    • This is highly dependent on the speed of your internet connection. Times can vary from less than 30 seconds to five minutes per chip for most high-speed connections. In general, it will take less than one minute to set up a download session for an unlimited number of files, unlike the frequent interventions needed to manually copy files from CDs. In addition, you can have extended download sessions for large numbers of chips going on in the background constantly with almost no impact to your computer's performance. If there is any interruption in your internet connection during a download you will not lose files that were transferred before the interruption.
  • How many PCR cycles are required in the preparation of the TruSight Myeloid libraries?
    • The TruSight Myeloid library preparation requires 27 cycles of PCR.
  • How many SNPs are on the Infinium OncoArray-500K?
    • The OncoArray-500K contains 499K attempted bead types. It includes a combination of SNPs from the HumanOmniExpress array and novel SNPs provided by the GAME-ON consortium.
  • How many SNPs can be added to the HumanHap550 Plus Genotyping BeadChip?
    • A maximum of 121,600 bead types (attempted SNPs) can be added to the HumanHap550 Genotyping BeadChip.
  • How many TruSeq Amplicon Cancer Panel samples do you recommend that I pool together to sequence on the MiSeq?
    • For 300 cycle MiSeq v2 kits, we recommend that up to 60 samples per MiSeq run are pooled. When this guidance is followed, >1000x mean coverage per amplicon per sample should be achieved in runs with 15M clusters passing filter. The new MiSeq v3 kits can deliver >25M clusters passing filter and can accommodate 96 cancer panel samples per MiSeq run to achieve the same >1000x mean depth of coverage.
  • How many TruSight One reactions are included in each kit? What are the minimum and maximum numbers of samples that I can multiplex per enrichment in my TruSight One enrichment pools?
    • The TruSight One 9-sample kit provides sufficient reagents to prepare a maximum of three (3) enrichment reactions of up-to 3-pooled samples per enrichment.  The TruSight One 36-sample kits provide sufficient reagents to prepare a maximum of three (3) enrichment reactions of up-to 12-pooled samples per reaction. Single sample enrichments are possible, but the reagent volumes are optimized for preparing three sets of pooled sample libraries at either the 3- or 12-plex levels mentioned previously.  

  • How many additional cycles of SBS reagents do I need to calculate into the run for sequencing dual-indexed libraries?
    • For dual index paired-end runs, there are 23 additional cycles (index & chemistry only).
      For dual-index single-read runs, there are 16 additional cycles of indexing.
      For information about the number of SBS kits required on the HiSeq, HiScanSQ, or GAIIx, see the user guide for your instrument guide.

  • How many amplicons can be processed at once?
    • Currently, TruSeq Custom Amplicon supports up to 1536 amplicons in a single reaction. The number of amplicons in the multiplex reaction will vary based on the custom design and can range from 16-1536 amplicons. If more amplicons are desired (e.g. 3072), two reactions can be used with the same genomic DNA sample input. A single user can easily prepare upwards of ~150,000 sequencer-ready amplicons in a single day (96 samples x 1,536 amplicons).
  • How many barcode plates do I need to run to finish my TruSeq Synthetic Long-Read DNA genome?
    • The Synthetic Long-Read application generates at least 600 MB of synthetic long reads (>1.5 kb). The number of barcode plates prepared depends on the target genome size and the depth of the existing build. A reasonable starting point might be 5-10x genome coverage of synthetic long reads for genome finishing.
  • How many bases constitute a region under a peak?
    • See the GenomeStudio ChIP Sequencing Module User Guide available in the GenomeStudio Portal for information.
  • How many components can I expect with my order?
    • Orders will receive five items: four reagent boxes including the MAP, and one package containing arrays.
  • How many cycles are required for template generation?
    • Template generation, the process of identifying defining cluster positions over the entire flow cell surface, is performed during the first five cycles of the sequencing run. To detect a cluster during template generation, there must be at least one base other than G in the first five cycles.
  • How many cycles can I perform with the MiSeq Reagent Kit?
    • Illumina offers a variety of kit sizes ranging from a 50-cycle kit to a 600-cycle kit. MiSeq Reagent Kit v3 is available in sizes of 150 cycles and 600 cycles. The MiSeq Reagent Kit v2 is available in sizes of 50 cycles, 300 cycles, and 500 cycles. For more information, see the MiSeq Reagent Kit support page .
  • How many cycles do MiSeq v3 kits support?
    • MiSeq v3 kits are kitted for 150 cycles and 600 cycles.

  • How many cycles does the NextSeq 500 support?
    • The NextSeq 500 can perform up to a 150-cycle paired-end run (2 x 150) using available NextSeq 500 kits. Kits are available in sizes of 300 cycles, 150 cycles, and 75 cycles, and each kit includes additional cycles for index reads. For more information, see the NextSeq 500 Kit Reference Guide.

  • How many cycles of sequencing are possible with SBS kits?
    • HiSeq v4 SBS kits:

      • The 250-cycle kit contains enough reagents for 275 cycles.
      • The 50-cycle kit contains enough reagents for 75 cycles.

      TruSeq Rapid SBS kits:

      • The 200-cycle kit contains reagents for 225 total cycles.
      • The 50-cycle kit contains reagents for 74 total cycles.

      TruSeq v3 SBS kits:

      • The 200-cycle kit contains reagents for 209 total cycles.
      • The 50-cycle kit contains reagents for 58 total cycles.
  • How many cycles of sequencing are possible with SBS kits?
    • HiSeq v4 SBS kits:

      • The 250-cycle kit contains enough reagents for 275 cycles.
      • The 50-cycle kit contains enough reagents for 75 cycles.

      TruSeq v3 SBS kits:

      • The 200-cycle kit contains enough reagents for 209 cycles.
      • The 50-cycle kit contains enough reagents for 58 cycles.
  • How many cycles of sequencing do small RNA libraries need?
    • This depends on the aim of the experiment. For expression profiling projects, we do not recommend longer than 36 cycles of sequencing. The small RNA molecules are normally 15–30 bases long, and sequencing beyond this point only sequences the adapter. Eighteen cycles may be sufficient. For discovery projects, it may be worthwhile to do 40–50 cycles. This will sequence into the adapter, but the adapter sequence will be long enough to be unambiguously identified and removed even on longer small RNA molecules.

  • How many cycles should be used during the Index Read for single-indexed libraries?
    • Index reads for single-read libraries use seven cycles reads. Illumina does not support six cycle index reads for single-indexed libraries.

  • How many different libraries can be sequenced on one NextSeq 500 flow cell?
    • Libraries are transferred onto the flow cell from a single reservoir on the NextSeq 500 reagent cartridge, reservoir #10. Therefore, any libraries that can be pooled can be sequenced together on one flow cell.
  • How many different species can the MiSeq metagenomics workflow identify?
    • The MiSeq metagenomics workflow identifies genera, not species, and can identify 1311 different genera.
  • How many empty ports are available on the reagent cartridge?
    • Three reservoirs on the MiSeq reagent cartridge are available for user-supplied custom primers. For more information, see Using Custom Primers on the MiSeq (part # 15041638 ).
  • How many flow cells can I run on the HiSeq?
    • The HiSeq 2000 is a dual flow cell system, which allows you to run two flow cells simultaneously. The HiSeq 1000 is a single flow cell system.
  • How many genes are targeted by the Cancer Panel?
    • Mutational hotspots from over 48 genes associated with cancer are targeted with 212 amplicons.
  • How many indexes are included in each TruSeq RNA Access Library Prep Kit?
    • The TruSeq RNA Access Library Prep Kit is available in a Set A and a Set B, each containing 12 indexes. When used together, sets A and B provide a total of 24 unique indexes.
  • How many indices are available for the TruSeq ChIP Sample Prep Kit?
    • There are a total of 24 unique indices: 12 in Set A and 12 in Set B.

  • How many indices are available with the TruSeq LT and HT kits?
    • LT kits: 12 unique single indices in Set A and 12 unique single indices in Set B

      HT kit: 96 unique dual indices

  • How many lanes are on a NextSeq 500 flow cell?
    • The NextSeq 500 flow cell contains four physical lanes. However, libraries are loaded onto the flow cell from a single reservoir. You can sequence one library or multiple pooled libraries on one flow cell.

  • How many lanes does the MiSeq flow cell have?
    • The MiSeq flow cell is a single-lane flow cell.

  • How many reactions are included in each Nextera Rapid Capture Enrichment Kit?
    • Samples

      Enrichment Reactions

      Plexity

      8

      8

      1

      24

      8

      3

      48

      8

      6

      72

      8

      9

      24

      2

      12

      48

      4

      12

      96

      8

      12

      288

      24

      12

  • How many reads are needed per sample for small RNA sequencing?
    • This depends on the application. For expression profiling, 1–2M mapped reads is a generally accepted range. For discovery applications, an increase to 2–5M reads may be considered.    
  • How many reads can I expect from an optimally clustered MiSeq flow cell?
    • You can expect about 15 million reads passing filter.
  • How many runs can I save in the software?
    • You are unlikely to exceed the storage capacity of the provided computer. Eco run files are small (~200–500 kb for a 40-cycle run), while the computer hard drive is 213 GB.
  • How many samples are there per index pair in one TruSeq HT sample prep kit?
    • Because each well of the 96-well HT adapter plate is single-use only, only one sample per index pair can be generated.

  • How many samples can I load into KaryoStudio?
  • How many samples can I prepare at the same time with TruSeq Targeted RNA Expression?
    • Illumina recommends preparation of 16-96 samples at a time. Smaller batch sizes are supported, as long as reagents are not freeze-thawed more than five times. Larger batch sizes may be performed by more experienced users, taking care that the OB1 beads do not dry during purification steps.
  • How many samples can I process through the Nextera Rapid Capture Enrichment protocol at one time?
    • 1 to 96 samples can be processed at a time through the Nextera Rapid Capture Enrichment workflow. When processing fewer than the maximum number of samples per kit (e.g., 8, 24, 48, 96, 288) Illumina recommends storing aliquots of reagents for subsequent use instead of repeated thawing and freezing of the same reagent tubes.
  • How many samples can I sequence at the same time with TruSeq Targeted RNA Expression?
    • This is dependent on the experiment plexity and MiSeq version. Illumina recommends using the TruSeq Targeted RNA Calculator to determine the number of samples that should be pooled together to achieve the depth of coverage required for your assay. Refer to the TruSeq Targeted RNA Expression tech note for information on how to design an experiment and determine the appropriate number of samples to be run on a flow cell.

  • How many samples can be generated per TruSeq HT sample prep kit?
    • TruSeq HT sample prep kits support 96 sample preps. It is not recommended that the adapter plate undergo more than 4 freeze/thaw cycles.  When less than the full set of 96 libraries are pooled and sequenced, it is important that libraries with compatible index combinations are used in the index pool. Please refer to the user guide for your TruSeq HT sample prep kit for more information.

  • How many samples can be processed per TruSeq ChIP Sample Prep Kit?
    • Each TruSeq ChIP Sample Prep kit contains enough reagents and adapters to process 48 samples. Set A and Set B each contain 12 unique indexes, with enough of each index sufficient for 8 individual samples.

  • How many samples can be processed per TruSeq DNA PCR-Free LT and HT Sample Prep Kit?
    • The TruSeq DNA PCR-Free LT kits contain sufficient reagents for 24 samples and the TruSeq DNA PCR-Free HT kit contains reagents for 96 samples. Illumina recommends using the LT kit if processing less than 24 samples at a time and the HT kit if processing more than 24 samples. Both the LT kit and HT kit can be used with either the Low-Sample (LS) or High-Sample (HS) protocols.
  • How many samples can be processed per TruSeq Nano DNA LT and HT Sample Prep Kit?
    • The TruSeq Nano DNA LT Sample Prep kits contain sufficient reagents for 24 samples and the TruSeq Nano DNA HT Sample Prep Kit contains reagents for 96 samples. Illumina recommends using the LT kit if processing less than 24 samples at a time and the HT kit if processing more than 24 samples. Both the LT kit and HT kit can be used with either the Low-Sample (LS) or High-Sample (HS) protocols.
  • How many samples can be processed per TruSeq Sample Prep v1, v2 or LT Kit?
    • TruSeq DNA and RNA Sample Prep kits - Set A

      • V1 - Contains enough reagents and adapters to process 48 samples. Each kit contains 6 unique indexes, each index sufficient for 8 individual samples. These indexes were optimized for lower plexity multiplexing of DNA samples. Indexes: 2, 4, 5, 6, 7, 12
      • V2/LT - Contains enough reagents and adapters to process 48 samples. Each kit contains 12 unique indexes, each index sufficient for 8 individual samples. Indexes: 2, 4, 5, 6, 7, 12, 20, 21, 22, 23, 25, 27

      TruSeq DNA and RNA Sample Prep kits - Set B

      • V1 - Contains enough reagents and adapters to process 48 samples. Each kit contains 6 unique indexes, each index sufficient for 8 individual samples. Supplemental kit for use with Optimized Kit. Indexes: 1, 3, 8, 9, 10, 11
      • V2./LT - Contains enough reagents and adapters to process 48 samples. Each kit contains 6 unique indexes, each index sufficient for 8 individual samples. Supplemental kit for use with Optimized Kit. Indexes: 1, 3, 8, 9, 10, 11, 13, 14, 15, 16, 18, 19

      While Illumina supplies additional indexed adapters in the TruSeq Sample Prep v2 kits, the fill volumes are kept at 8 samples to minimize disruption to the protocols.  The v2 kits are limited by other components to 48 samples per kit.  This also offers more flexibility for how customers use the indexed adapters.

  • How many samples can be processed per TruSeq Small RNA Sample Preparation Kit?
    • TruSeq Small RNA Sample Preparation Kits are ordered as a core box and an index box. Each core box contains enough reagents to process 24 DNA samples and each index box contains 12 unique indexes, with sufficient index for two individual samples.

  • How many samples can be run on one flow cell?
    • Flow cells are designed for single-use. All eight lanes must be used at the same time. They can be used for the same sample or for dif­ferent samples. You can run eight samples at a time without multiplexing. With multiplexing, you can increase throughput to up to 12 samples per lane or up to 96 samples per flow cell.
  • How many samples can be sequenced at once?
    • TruSeq Custom Amplicon has integrated sample barcodes that enable pooling of up to 96 samples per MiSeq run. However, the actual number of samples that can be pooled together per sequencing run depends on the number of amplicons and the desired depth of sequencing coverage. An online calculator is provided in DesignStudio to help with these calculations.
  • How many samples can be sequenced at the same time when performing the TruSight DNA Amplicon assay?
    • The TruSight DNA Amplicon has integrated sample barcodes that enable pooling of up to 96 samples per MiSeq run. However, the actual number of samples that should be pooled together per sequencing run depends on the number of amplicons and the desired depth of sequencing coverage. For TruSight Myeloid libraries, eight samples per MiSeq run with MiSeq v3 reagents should give the appropriate amplicon sequencing depth.
  • How many samples should I load per run when performing the TruSight Myeloid assay?
    • Illumina recommends the following to give > 95% of amplicons at > 500x coverage:
      • 8 samples per MiSeq v3 run
      • 4–5 samples per MiSeq v2 run
      • 42 samples per NextSeq 500 Mid Output run. Customers can also run fewer samples to achieve higher coverage economically.
      • 96 samples per NextSeq 500 high output run or HiSeq 2500 rapid run. (96 samples is the maximum per run due to the number of indexes available.)
  • How many samples should I run for clustering on custom Infinium content?
    • Illumina recommends that you run at least 100 samples including both replicates and trios.
  • How many swaths and tiles are on a two-lane rapid run flow cell?
    • Scanning and analysis of a two-lane rapid run flow cell creates two swaths per surface on two surfaces per lane. Each swath is divided into 16 tiles. For a two lane flow cell, there are a total of 128 tiles per flow cell.
  • How many swaths and tiles are on an eight-lane HiSeq v4 high output flow cell?
    • Scanning and analysis of a HiSeq v4 flow cell is performed in three swaths per surface on two surfaces per lane. Each swath is divided into 16 tiles. Therefore, an eight-lane flow cell contains 768 tiles per flow cell.
  • How many targets can I multiplex with NuPCR?
    • DesignStudio can design duplex asssays.
  • How many technical replicates should I run per sample with NuPCR?
    • It is good practice to use 3 technical replicates. Ultimately, it is the decision of the user to define what is appropriate for their experiments.
  • How many tiles are imaged on a NextSeq 500 flow cell?
    • There are two types of flow cells that can be sequenced on the NextSeq 500, the high-output flow cell and the mid-output flow cell. Both flow cells contain four lanes, but the lanes differ in width resulting in a different number of tiles.

      • Lanes on the high-output flow cell are imaged top and bottom in three swaths, or columns, for a total of 864 tiles.
      • Lanes on the mid-output flow cell are imaged top and bottom in one swath for a total of 288 tiles.

      For more information, see the NextSeq 500 System User Guide.

  • How many times can I add additional content to a previously ordered Nextera Rapid Capture Custom Enrichment panel?
    • Additional target regions can be added to exiting content panels as many times as you like.
  • How many transcripts can I target with TruSeq Targeted RNA Expression?
    • Your order can contain 12-1000 probes.
  • How many uses can you obtain from the index kits?
    • The index kits are good for four uses. The 96-index kit has enough reagents for 384 samples; the 24-index kit has enough reagents for 96 samples. Additional caps are included for the tubes to replace after each use in order to prevent cross-contamination.
  • How much DNA can be targeted with this product?
    • TruSeq Custom Amplicon enables targeting of over ~650 kb of cumulative DNA sequence (1536 amplicons x 425 bp each = ~650 kb).
  • How much DNA is required for bisulfite conversion?
    • Single use, manual process: > 500 ng, Single use, automated process: > 1,000 ng, Multi use, manual process: 2,000 ng, Multi use, automated process: 2,000 ng
  • How much DNA is required for the GoldenGate Assay for Methylation?
    • 250 ng, 5 ul at 50 ng/ul.
  • How much DNA is required for the Infinium Methylation Assay?
    • Going into the bisulfite conversion, at least 500 ng is required for the manual protocol, and at least 1000 ng is required for the automated protocol.

  • How much DNA is required to load a flow cell lane for bridge PCR?
    • If possible, start the sample prep with 1–5 μg of DNA, although 1–2 μg is enough for many flow cells.
  • How much additional time will be added to the current sequencing run time on HiSeq to support dual indexing?
    • When using v3 SBS reagents and the v3 flow cell on HiSeq, each additional index cycle is approximately 53 minutes per cycle (with 16 total indexing cycles) plus ~2 hours for the seven chemistry-only cycles for the PE workflow, resulting in ~16 hours of additional time for dual indexing on HiSeq.
  • How much genomic content can I target in my Nextera Rapid Capture Custom Enrichment project?
    • Nextera Rapid Capture Custom Enrichment is available for designs that include 2000–67,000 total probes. Depending on probe density within the designed regions, this represents about 0.5–15 Mb of genomic content.
  • How much input RNA do I need for TruSeq Targeted RNA Expression?
    • The protocol is optimized for 50 ng of total RNA. For degraded samples with ≥ 200 bp fragments, Illumina recommends the RNA input be optimized for 100–200 ng, depending on the RNA quality.
  • How much input genomic DNA is required for the Nextera rapid Capture Enrichment workflow?
    • 50 ng of input genomic DNA is required. It is important to accurately quantify the input genomic DNA to generate a high quality library of the correct size.  Illumina highly recommends using a fluorometric-based quantification method for the input genomic DNA. For more information, see the DNA Input Recommendations section of the Nextera Rapid Capture Enrichment Guide .
  • How much input genomic DNA is required for the TruSight One workflow?
    • 50 ng of input genomic DNA is required. It is important to quantify the input genomic DNA to generate a high-quality library of the correct size.  Illumina highly recommends using a fluorometric-based quantification method for the input genomic DNA. For more information, see the DNA Input Recommendations section of the TruSight One Library Prep Guide .
  • How much starting material does the ChIP-Seq Sample Preparation Kit require?
    • The ChIP-Seq Sample Preparation Kit is designed to use 10 ng of ChIP-enriched DNA. Many users have reported that using at least 30 ng of DNA makes the protocol simpler and more repeatable.

  • How much template does the MiSeq use?
    • The MiSeq uses a total of 500 µl of denatured and diluted template for priming and clustering. However, 600 µl of template is recommended to avoid air bubbles and accommodate for instrument sipper depth. This volume does not vary for different workflows.
  • How much time do typical HiSeq v4 runs take?
  • How much time do typical Rapid runs take?
    • Read Length Estimated Rapid Run Time (Hrs)*
      1 x 50 bp no index 9
      1 x 50 bp dual index 11
      2 x 100 bp no index 27
      2 x 100 bp dual index 30
      2 x 150 bp no index 40
      2 x 50 1bp dual index 43
      *Systems with SN < 7000895 will require additional time
  • How much total RNA input is required for TruSeq RNA Access Library Prep?
    • Illumina recommends 10 ng of high-quality universal human reference total RNA as input. If starting with FFPE RNA, the sample input amount is based on sample quality. Illumina recommends using the percentage of RNA fragments > 200 nt fragment distribution value (DV200 ) as a reliable determinant of FFPE RNA quality.

      Quality

      DV200

      Input Requirement Per Reaction

      High

      > 70%

      20 ng

      Medium

      50-70%

      20-40 ng

      Low

      30-50%

      40-100 ng

      Too degraded

      < 30%

      Not recommended

      For more information, see the Evaluating RNA Quality from FFPE Samples tech note.
  • How often do you update your internal database?
    • Updates coincide with dbSNP releases.
  • How often is the annotation updated?
    • This depends on updates to the Sanger miRNA database, http://microrna.sanger.ac.uk/sequences/. We plan to update the annotation as needed.
  • How often is the content updated?
    • We update content as often as warranted by Sanger miRBase. Our current content covers Sanger v12.
  • How should ChIP DNA be quantitated?
    • We recommend quantitating ChIP DNA with the most sensitive method available, taking into account the low amount of DNA that is often available. Fluorescent dye-binding assays such as PicoGreen or Qubit offer high accuracy, assuming precise pipetting. The Bioanalyzer and High Sensitivity chips can also give good quantitation, and allow you to check the size and purity of your sample.

  • How should I measure the final mRNA-Seq library?
    • We recommend using the Agilent 2100 Bioanalyzer to estimate final product concentration and check the size of the library. We do not recommend using only NanoDrop because its concentration estimate includes primers and other small nucleic acids.
  • How should I quantify my input samples for NuPCR?
    • Typically input cDNA is quantified by OD260 or by fluorecent intercalating dyes. Starting RNA can be evaluated using an Agilent Bioanalyzer to examine quantity and integrity.
  • How should I quantitate my DNA before GoldenGate genotyping?
    • We recommend using a method of direct quantitation specific to double-stranded DNA (dsDNA), such as with the PicoGreen reagent. Alternative indirect methods such as UV spectrometry/NanoDrop can incorrectly report the concentration of dsDNA in your sample when single-stranded DNA, oligonucleotides, RNA, and/or proteins carried over from DNA extraction are present in solution.
  • How should I send DNA samples to Illumina for genotyping?
    • We will provide to you barcoded 96-well plates for you to ship samples to us along with a step-by-step preparation protocol.
  • How should I specify the splice junction set?
    • As of CASAVA v1.7, eland_rna uses the refFlat.txt.gz or seq_gene.md.gz file to generate the splice junction set automatically.

  • How should I test the quality of RNA as the TruSeq RNA starting material?
    • Illumina recommends that you check total RNA integrity following isolation using an Agilent Technologies 2100 Bioanalyzer with an RNA Integrity Number (RIN) value greater than or equal to eight.

  • How should the Resuspension Buffer (RSB) be stored?
    • Resuspension Buffer should be stored at -15 to -25ºC when first received. After the initial thaw, the reagent can be stored at 2 to 8ºC for use throughout the protocol.
  • How was the default known region list generated?
    • The known region list covers 244 known regions and was generated by compiling information from multiple sources including publications, databases, and public information available on which regions are typically examined in cytogenetics labs.
  • How well do SNP genotype calls from the Infinium Assays compare to those called from the GoldenGate Assay?
    • Although we have not tested this specifically, the expectation is that the conversion efficiency will be high.
  • How well do SNP genotypes called using the GoldenGate Assay compare to those called using the Infinium Assay?
    • Over 65% of the SNPs genotyped by the HapMap project phase I were done using the GoldenGate Assay. The SNP content on the HumanHap300 Genotyping BeadChip was chosen from HapMap phase I validated SNPs. Our scientists found a high conversion rate of HapMap-validated SNPs.
  • How were the SNPs selected?
    • The OncoArray consortium chose the selected SNPs specifically to develop a broad-use array platform that provides a detailed querying of:

      • Common cancers, including breast, prostate, ovarian, colorectal, and lung cancer
      • Validated cancer-related SNPs, as well as genetic association analysis of pharmacogenetic variation
      • GWAS backbone that allows new discoveries for all cancers
  • How will I know if I have the most up-to-date documentation about the Eco system?
    • Check http://www.illumina.com/ecoqpcr regularly to find out about new Eco system developments and applications. In addition, read the Illumina monthly customer newsletter, Illuminotes, to be promptly notified of any changes to systems, applications, or documentation.

  • How will I know what are the best index combinations for my sample prep?
    • The Illumina Experiment Manager (IEM) will notify you if improper combinations are used when creating a sample sheet for use with CASAVA, so it is highly recommended to create your sample sheet prior to performing sample prep/pooling. There are also low plexity pooling guidelines in the Nextera, TruSeq DNA, and TruSeq RNA sample preparation guides. Always pool samples with valid index combinations to avoid image registration failures.
  • How will I know what are the best index combinations?
    • The Illumina Experiment Manager (IEM) will notify you if improper combinations are used when creating a sample sheet, so it is highly recommended that you create your sample sheet prior to performing sample prep/pooling. There are also low plexity pooling guidelines in the Nextera DNA Sample Preparation User Guide. Always pool samples with valid index combination to avoid image registration failures.
  • How will my TruSeq controls be shown for multiplexed samples?
    • You can filter by Index in SAV (Sequencing Analysis Viewer) for indexed runs.

  • How would I enable analysis of both single and dual index samples in the same flow cell using CASAVA 1.8.2?
    • If dual-index libraries are combined with single-index libraries on the same flow cell, single-indexed libraries will have sequence that can be ignored for the second index read. For this combination, you need to create two sample sheets: one sample sheet for lanes containing single-index libraries and another sample sheet for lanes containing dual-index libraries. Run CASAVA separately with the appropriate --use-bases-mask command for the index reads to demultiplex appropriately. See Sequencing Mixed Libraries on a HiSeq or GA Flow Cell.

  • I am currently finishing a project using MiSeq v2 reagents. Can I upgrade my software to MCS v2.3 and still finish my project with v2 reagents?
    • Yes. The new MiSeq software package is backward compatible with v2 reagents. Using the RFID feature, the MiSeq automatically recognizes which kit version is loaded for the run and chooses the appropriate Q-table. There are no changes to v2 workflows.

  • I am currently running traditional PCR; can I use the same template, primers, and reagents to run Real-Time PCR?
    • In some cases it is possible to convert existing traditional PCR assays into Real-Time PCR assays, with a few considerations around primer design and master mix. Primer design is one of the first considerations for converting a traditional PCR assay. Real-Time PCR is most efficient with relatively short amplicon lengths, in the range of 50 to 150 bp. Longer products can be used if the cycling conditions are changed to accommodate longer extension times, but you should avoid products longer than 300 bp. In some cases it might be possible to design a TaqMan probe to hybridize between the two existing PCR primers. If not, you can use SYBR Green I for detection. (See FAQs What are the advantages and disadvantages of TaqMan and SYBR Green I chemistries? and Are SYBR Green I Real-Time PCR assays less specific than TaqMan probe assays? for more information on TaqMan and SYBR Green I.)

      The master mix is another consideration when converting a traditional PCR assay into a Real-Time PCR assay. If a TaqMan probe can be designed, you might be able to use the same master mix that was used for the traditional PCR assay. If a TaqMan probe cannot be designed, you should add SYBR Green I to the master mix. In either case, a certain amount of optimization may be needed to obtain good Real-Time PCR results.

  • I am currently using the TruSeq DNA Sample Prep Kit. Should I switch to the TruSeq DNA PCR-Free Sample Prep Kit or the TruSeq Nano DNA Sample Prep Kit?
    • Illumina will be discontinuing the TruSeq DNA Sample Prep kits, with orders for these kits being accepted through December 29, 2013. The Sample Preparation: Kit Selector can assist in choosing whether the TruSeq DNA PCR-Free Sample Prep or TruSeq Nano DNA Sample Prep kit best fits your needs. In summary, the kit selected depends on available sample input amounts and the quality of data.

      • The TruSeq DNA PCR-Free Sample Prep Kit requires 1 µg of gDNA, while the TruSeq Nano DNA Sample Prep Kit requires 100 ng of gDNA.
      • The TruSeq DNA PCR-Free kit delivers the utmost data quality by eliminating PCR-induced biases, specifically, greater coverage of hard to reach regions, such as fosmid "difficult" promoters.
      • The TruSeq Nano DNA kit generates premier data quality, superior to TruSeq DNA, however it does have some PCR-induced bias and PCR duplicates, and cannot cover "difficult" promoters as well as TruSeq DNA PCR-Free.
      • TruSeq Nano DNA final libraries can be quantified using either qPCR or a fluorometric method using dsDNA binding dyes such as Qubit or Picogreen, while TruSeq DNA PCR-Free final libraries can only be quantified using qPCR.
  • I am getting an error when running the Upload Test Application. What should I do now?
    • Please save a screenshot of the error, along with any logs that were created from the Test Application. If the Test Application has completed, these files are saved in the same folder on your computer that the program is in. Contact TechSupport@illumina.com.
  • I am used to filling a standard 96-well plate with my samples; how can I tell which well my sample is pipetted into?
    • The Eco sample loading dock provides a backlight that highlights the content of the wells. The light increases the contrast of the alpha numeric sample locators and helps you monitor sample loading. The dock has an adjustable foot that lets you angle the plate for optimal visibility if needed; for example, when loading under a laminar flow hood.

  • I am using a kit that includes an internal control, and so I need to select two channels for reading. Where do I select the channels?
    • On the Plate Layout tab, click the Assays button to open the Assays dialog box. Add two assays, one for the target and another for the IPC. Assign reporter dyes from different channels to each assay.

      Regardless of the plate setup, the Eco system acquires data in all four channels for all 48 wells. The dyes enable you to focus on the desired information during analysis.

  • I broke the gel when opening it during TruSeq Synthetic Long-Read DNA library prep. Can I still use it?
    • Yes, as long as you are still able to accurately determine the position of the 10 kb and 8 kb bands of the ladder in relation to the sample.
  • I cannot find a Report Wizard in the PC Module. How can I create reports from my data?
    • Data can be exported directly from the Samples Table, SNP Table, and Full Data Table for downstream analysis. Mark the columns and rows you wish to export and click the icon for "Export displayed data to file" to save selected table content in *.txt or *.csv format.

  • I can’t see a reference sequence in the IGV (Illumina Genome Viewer) or ICB (Illumina Chromosome Browser). How can I display a reference sequence?
    • See Chapter 5 of the GenomeStudio 2008.1 Framework User Guide, available on iCom and in the GenomeStudio Portal.

  • I clicked the 'Download Checked Samples' option, and it completed the download. Why can't I find the genotyping results?
    • When you click the Download Checked Samples button only functions to generate the genotype data. You must also click the Generate Report button to generate the report and save it to your computer.
  • I created a query using the 'Edit Query' button, and then clicked 'OK'. Why didn't my list of samples change?
    • The Edit Query function only sets up the query to be performed. After the query is set, click Perform Query to update your results.
  • I do not have GenomeStudio installed on my computer, but would like to work with the PC Module. Do I need to install GenomeStudio prior to installing the PC Module?
    • Yes, please download the GenomeStudio 2011.1 installer from the Illumina website. It is sufficient to install the GenomeStudio Framework by clicking the respective box in the install wizard, which does not require a license key. It is not required to install the GenomeStudio Genotyping Module on the same computer on which the PC Module is installed. However, the polyploid workflow does require generating a genotyping project in the GenomeStudio Genotyping Module prior to taking the data to the PC module for polyploidy clustering. 

  • I don't have an iCom account. Can I still access the Illumina iControlDB to download genotypes?
    • No. Access to downloading the genotypes is only available with an iCom username and password. Illumina customers can sign up for an iCom account by visiting http://icom.illumina.com
  • I get the following error message, what should I do? ERROR: previous run [13_01_39_04_09_08] didn't finish
    • One option is to clean all your data and run your command CMD again ˜/ CMD --target=allClean ˜/ CMD
  • I get the following error message, what should I do? ERROR: reference sequence /data/runs/genomes_human/c1.fa does not exists
    •  If you run into the ERROR: reference sequence ⁄data⁄runs⁄genomes_human⁄c1.fa does not exist, there is a good chance that you are not using files provided by Illumina.

      Assuming that you have specified the reference genome to be: --refSequences=/data/runs/genomes_human and genome size file: -g conf/human_rna_size.xml you have three options:

      1. Use the human reference genome provided by Illumina. This is preferable since the GenomeStudio Software assumes human reference genome provided by Illumina.

      2. Change the names of your fasta files in ⁄data⁄runs⁄genomes_human to match the names in human_rna_size.xml.

      3. Instead of using conf⁄human_rna_size.xml, use genome_sizes.xml produced by Pipeline.

      NOTE: For CASAVA for RNA Sequencing all reference sequences and genome_size files are provided by Illumina.

  • I got a warning message in HCS about the ARM9BoardSerialPort. What does this mean and what should I do?
    • The warning message "ARM9BoardSerialPort (ARM9CHEM): timed out waiting" indicates that an ARM9 communication time out has occurred. The ARM9 board is one of many components that communicate between the HiSeq and instrument computer.  Messages related to an ARM9 time out are not necessarily indicative of a hardware issue, and do not impact the run or data quality.

      If this message appears repeatedly, perform a normal stop on the current run, shut down the HCS/RTA software, and then power cycle the HiSeq and instrument computer to reestablish communication between the systems. Launch HCS and resume your run.  Continue to monitor your run to make sure that the issue is resolved. If it appears that the run data is affected, contact Illumina Technical Support for further assistance.

  • I got a warning message in HCS about the Tdi Scan. What does this mean and what should I do?
    • TDI Scan warning messages indicate an issue with image acquisition and storage; however, the system will automatically retry image capture to self-correct. TdiScan messages usually have no effect on the run other than slightly extended cycle times, and do not affect the run data as images are re-captured before continuing.

      In the rare event that the retry threshold is exceeded, one imaging swath is skipped for one cycle. If this message occurs frequently, contact Illumina Technical Support for assistance.

  • I have downloaded specific samples before, but now when I open the iControlDB - Client, it says “FALSE” in the Downloaded column for these samples. Why does this happen?
    • The TRUE and FALSE entries in the Downloaded field are only applicable to the current session. If you have downloaded samples in previous sessions, this will not be reflected in the current iControlDB - Client session.
  • I have uploaded my data, but it is not visible using the iControlDB download software. Was there a problem with the upload?
    • All uploaded data is initially deposited in a staging area until it is reviewed by Illumina personnel. Data is not released to iControlDB until it has undergone this review process, which may take up to 10 business days. Additionally, data will not be released if they do not pass our call rate threshold of 98%. If you still do not see your data after 10 business days, please contact techsupport@illumina.com for further investigation.

  • I received a warning message “need at least 750 GB space in local sever” when starting a run. Is the run folder on the local computer 750 GB in size?
    • This amount of free space is required at the beginning of a run. The system assumes that data is transferred to the network copy of the run folder in real time. Therefore, 750 GB is the safe level to start a run. The software assumes that the run copies and deletes the files as they are processed, and that the connection to the network server can keep up with file transfer.

  • I see a 130-135 bp band on my final ChIP-Seq library. What is this?
    • Although the Illumina adapters are designed to reduce adapter-adapter ligation, very low amounts of starting material can result in high adapter-insert ratios during ligation. This promotes formation of adapter dimers and occasional adapter concatamers. These concatamers often take the form of amplified trimers in various configurations. They can be removed by careful size selection, or by repeating the ligation with more input DNA or less adapter. The amount of adapter added to the ligation can be titrated by additional dilution (i.e. a 20× or 50× dilution, rather than the 10× dilution described in the protocol).

  • I see a design gap in my Nextera Rapid Capture Custom Enrichment target region. Is there anything that I can do about this?
    • Design gaps can occur due to complex genomic regions, low probe specificity, skewed GC content, and other technical factors inherent to probe based enrichment. The gap can be resubmitted for design as a New Target Region using the gap start and stop chromosome coordinates.
  • I see lower coverage of GC regions than I was expecting with the TruSeq Nano DNA Sample Prep protocol. What could be a factor?
    • Coverage of GC regions can be impacted by the model, settings, and performance of the thermal cycler used. Illumina has validated the Bio-Rad DNA Engine Tetrad 2, the Bio-Rad S1000, and the MJ Research PTC-225 DNA Engine Tetrad. Other thermal cyclers may differ in their performance across the genome.
  • I tried to cluster a set of SNPs, but no clusters are visible in the SNP Graph. What went wrong?
    • SNPs are only clustered for samples selected in the Samples Table (marked in blue). If no samples are selected in the Samples Table, SNPs are clustered for all samples in the Samples Table (except non-excluded samples). Thus, if you wish to cluster SNPs for all your non-excluded samples please make sure that no samples are selected in the Samples Table at the time of clustering (e.g. by clicking onto an area in the SNP graph).

  • I uploaded my data, and it has been reviewed and released by Illumina. Why are some of my samples missing?
    • We review the data for many criteria before releasing to the database. Any samples that do not pass our call rate criteria are not included in the released data. Additionally, there may be user-entered information (e.g., ethnicity or positive phenotype) that is unclear or not compliant with the Health Insurance Portability and Accountability Act (HIPAA). These samples will not be included in the released data.

  • I'm getting error messages running CASAVA. What do they mean?
    • Please send an email to Illumina Technical Support (techsupport@illumina.com ) that includes the nohup output file and the command line you used. One of our bioinformatics specialists will respond.
  • If I am doing low levels of pooling with TruSeq sample prep kits, what considerations do I need to make for the index selections?
    • TruSeq kits support many low plex pooling options across the entire plate. Some of these combinations are outlined in the Pooling Guidelines section of the appropriate TruSeq sample preparation guide. Customers can also design their own color-balanced pools, but we highly recommend that the Illumina Experiment Manager be used to check the color balance of user-designed pools
  • If I am running a software version older than HCS 1.4.8, do I need to serially upgrade to 1.4.8 prior to installing HCS 1.5?
    • You can upgrade directly to HCS1.5/RTA1.13 from HCS1.4/RTA1.12. If you are running an older version of HCS, please contact Illumina Technical Support for assistance in upgrading.

  • If I choose to design my TruSeq Custom Amplicon project with SNP avoidance turned off what effect will it have on my data?
    • If an upstream or downstream amplicon primer region overlaps an actual variant in the input genomic DNA, the assay may experience reduced specificity and/or uniformity relative to other amplicons in the multiplexed reaction.
  • If I continue to use VariantStudio v2.1, do I get the updated Illumina Annotation Service?
    • No. VariantStudio v2.1 is programmed to use annotations from the previous version of the Illumina Annotation Service. It does not connect to the new version that is available with VariantStudio v2.2.  If you want to use the new version of the Illumina Annotation Service, use VariantStudio v2.2 and reannotate any samples in projects from VariantStudio v2.1.

  • If I do not design my NuPCR assays to be multiplexed in DesignStudio, can I multiplex them later?
    • You can attempt to combine individual assays designed with separate dyes.  However these assays will not be checked for compatiblity.  We recommend using DesignStudio to create your duplex assays for a greater chance of success.
  • If I have added additional information in auxiliary columns in my BeadStudio project, will this be included in the released data?
    • No. Only information that is contained in the standard phenotype information columns will be available from iControlDB during the download process.
  • If I have my own HGMD Professional license or if I want to use the public HGMD version, can I import HGMD annotations into VariantStudio?
    • You can import external sources of annotations into VariantStudio using the custom annotation feature. Check your HGMD Professional or HGMD public version license terms to make sure that such use is permitted.

  • If I reannotate my samples from the VariantStudio v2.1 projects in VariantStudio v2.2, can I keep both sets of annotations?
    • No. If samples are reannotated in VariantStudio v2.2, all annotations for variants are replaced with the annotations from the newer version of  the Illumina Annotation Service. If you do not want to lose previous annotations for these samples, create a project for the samples that you want to preserve with the old annotations.

  • If I run two flow cells at the same time, do they need to be identical in setup?
    • No. You can start each flow cell independently from the other. Each flow cell can have a different number of reads and cycles.

  • If I send my data to BaseSpace, what analysis options do I have?
    • You can use Illumina’s BaseSpace Core Apps to analysis data in BaseSpace. Available apps include BWA and Isaac WGS, RNA Seq, Enrichment, and Tumor-Normal analysis. Please see https://basespace.illumina.com/apps for more details, including descriptions of each app.
  • If I use BaseSpace for run monitoring only, what files are sent to BaseSpace?
    • The files that are sent to BaseSpace are the InterOp folder, RunInfo.xml file, and RunParameters.xml file.
  • If my NuPCR assay is not working how can I optimize the assay?
    • NuPCR assays are already bioinformatically screened for a consistent melting temperature and NuPCR Master Mix. It should not be necessary to vary PCR conditions. Changing conditions may result in worse performance. 
  • If my NuPCR experiment failed on non-Eco qPCR instruments, can Illumina support the troubleshooting?
    • NuPCR is sold only through distributors. Contact your regional distributor for assistance and support with NuPCR.
  • In VariantStudio v2.2, can I choose to continue to use the previous annotation database version?
    • No. VariantStudio v2.2 is programmed to use annotations from the version of the Illumina Annotation Service that was updated for VariantStudio v2.2.

  • In the NuPCR assay why is the fluorescence measured at the annealing step rather than the extension step?
    • The  NuZyme complex is formed when Partzyme A and B combine with  the target sequence and the substrate oligo.  This only happens during the Annealing step of the PCR cycle so this is when fluorescence is measured.
  • In what format does MiSeq Reporter output aligned data?
    • MiSeq Reporter outputs aligned data in the BAM file format.
  • In what format will Illumina provide the final data at the end of the project?
    • We will provide you with compact discs containing the final data in standard comma-delineated text. For large genotyping studies, we will be happy to discuss custom formats with you.
  • Is DNA fragmentation (i.e., with Covaris) necessary?
    • No. The tagmentation step in the Nextera protocol eliminates the need for mechanical fragmentation/shearing.
  • Is GenomeStudio software supported on Windows Vista?
    • Yes, GenomeStudio is supported for Windows XP, Vista and Windows 7 (see Computing Requirements http://support.illumina.com/array/array_software/genomestudio/computing_requirements.ilmn ).
  • Is HCS v2.2 available for non-upgradeable systems?
    • No. HCS v2.2 is only available for instruments that are eligible for the HiSeq v4 upgrade.
  • Is Illumina data compatible with Bioconductor?
    • Yes, Illumina data is compatible with Bioconductor, a collection of R packages developed by researchers around the world and distributed for free.
  • Is MiSeq scalable?
    • MiSeq offers scalable throughput based on read length. Illumina continues to increase read lengths, imaging area, and cluster density with improved detection and resolution. For example, at launch the MiSeq performed up to 150-cycle paired-end runs (2 x 150 bp) with greater than five million reads passing filter. Currently, the MiSeq can perform up to 250-cycle paired-end runs (2 x 250 bp)  that generates 15 million reads passing filter. For more information, see the MiSeq Product Information Sheet.

  • Is NaOCl required for the instrument wash?
    • An instrument wash is performed automatically when the run completes. For this wash, a dilute solution of 0.03%–0.06% NaOCl is required. To prepare the automatic post-run wash, load 3 ml in reservoir #28 before loading the reagent cartridge. A manual wash is performed as needed from the NCS Welcome screen. As of the release of NCS v1.2, or later, the addition of NaOCl is no longer required with a manual wash. If you are using an earlier version of NCS, the software expects 3 ml 0.03%–0.06% NaOCl in position #28 of the reagent wash tray.
  • Is TruSeq RNA Sample Prep compatible with the Directional mRNA-Seq Sample Prep?
    • The Directional mRNA-Seq Sample Preparation Guide provides instructions to use the Illumina Small RNA adapters, which are specific to the 3' and 5' fragment end, to generate a stranded library with the original Illumina mRNA-Seq kit. The protocol has not been optimized for the TruSeq RNA Sample Prep Kit and Illumina does not recommend this use of the TruSeq RNA Sample Prep Kit.

  • Is TruSeq Targeted RNA Expression oligo design biased to the 3' end of mRNA?
    • No, DesignStudio assays target splice junction sites across the entire transcript. Additional designs are available for cSNPs or gene fusions. The following databases were used for assay designs:

      Species

      Database

      Human junction

      hg19 + refseqs transcript model

      Human cSNP

      dbSNP build 137, common SNPs ≥ 1% MAF

      Mouse junction

      mm9, mm10 + refseq transcript model

      Mouse cSNP

      dbSNP build 128 for mm9, dbSNP build 137 (common SNPs) for mm10

      Rat junction

      rn5,rn4

      Rat cSNP

      dbSNP build 125 for rn4

  • Is VariantStudio available as a command-line tool?
    • No. Illumina VariantStudio was is a point-and-click software application that enables variant data exploration, annotation, and filtering without requiring bioinformatics expertise.

  • Is a Bioanalyzer trace required to be run after enrichment to check the library size?
    • A post-Enrichment Bioanalyzer quality control step is optional. Running a Bioanalyzer trace can be a useful tool for troubleshooting lower than expected cluster densities by recalculating the post-Enrichment library concentrations with the returned library size estimate. If the average post-Enrichment library size is larger than expected, the actual loading concentration is lower than expected, resulting in lower raw cluster densities.
  • Is a PhiX control included in the TruSeq Cluster Kit?
    • No. The Illumina PhiX Control v3 is a separate product. The catalog number is: FC-110-3001.
  • Is a PhiX control lane necessary with ChIP-Seq libraries?
    • Yes. Illumina recommends using a PhiX control lane when sequencing ChIP-Seq libraries. Samples that contain genomes with high AT or GC content (less than 40% or greater than 60%) require a dedicated PhiX control lane for cross-talk and phasing calculations. For more information, see Using a PhiX Control for HiSeq Sequencing Runs .
  • Is a PhiX control lane necessary with small RNA libraries?
    • Many small RNA libraries do not require a PhiX control lane to generate accurate matrix and phasing estimates. This is especially true in the case of samples from organisms with balanced genomes (e.g. mammalian organisms). Libraries from organisms with unbalanced genomes, or libraries with a high proportion of reads from a single template (for example, adapter reads, or samples from a tissue that expresses a single small RNA at high levels), may require use of a control lane for matrix and phasing generation. In any case, a PhiX control lane acts as a positive control for run performance and allows error rate estimation.
  • Is a sample sheet required for TruSeq Synthetic Long-Read DNA library prep? If so, what application do I use in the Illumina Experiment Manager software?
    • A sample sheet is required prior to starting a sequencing run and enables the correct identification and processing of libraries after the sequencing run is streamed to BaseSpace. Use IEM v1.8 or later and select the TruSeq Synthetic Long-Read DNA kit and the HiSeq FASTQ Only application.
  • Is a sample sheet required on the NextSeq 500?
    • No. The NextSeq does not expect a sample sheet during run setup. When connected to BaseSpace, library and indexing information is entered on the BaseSpace Prep tab before the run, and the information is passed to the NextSeq. The available run names appear on the instrument screen during the run setup steps.
  • Is a sample sheet/library sheet optional or mandatory for sequencing runs and analysis?
    • For runs on the HiSeq, HiScanSQ, or GAIIx, creating and loading a sample sheet at the start of the run is optional. However, using a sample sheet allows you to view data shown on the indexing tab in the Sequencing Analysis Viewer (SAV) during the run. If you do not load a sample sheet at the start of a run in HCS, you will not be able to view indexing data in SAV. When analyzing indexed samples using CASAVA v1.8.2, a sample sheet is required. MiSeq runs require a sample sheet when setting up the run in MCS.

      Illumina recommends that you create the sample sheet using the Illumina Experiment Manager (IEM) prior to performing sample prep in order to confirm appropriate index combinations.

  • Is any validation performed on NuPCR assays before they shipped? Are multiplex assays experimentally tested to ensure performance before shipping?
    • NuPCR assays are bioinformatically screened using proprietary algorithms to identify successful primer sequences.
  • Is automation available for the GoldenGate Assay for Methylation protocol?
    • Yes, see the user guide for instructions on the automated protocol, robot use, and liquid-handling automation.
  • Is bisulfite sequencing compatible with the TruSeq Nano DNA Sample Prep Kit?
    • Only LT adapters are methylated and the polymerases used in the TruSeq kits are not appropriate for bisulfite applications.
  • Is ethidium bromide used in the gel purification step of mRNA-Seq library preparation?
    • Yes. You can add ethidium bromide or another DNA-visualizing fluorophore to the gel.
  • Is initial cycle indexing possible on the HiSeq?
    • No, the system does not support an initial cycle indexing method. To ensure the highest quality data, Illumina recommends and supports a separate indexing read for multiplexed samples.
  • Is it OK if I still see primers after the final Qia-Quick PCR cleanup?
    • Yes, this is OK. Their presence will not impact the performance of the assay.
  • Is it a problem to run low numbers of indices/pooled samples? What will happen if I run improper index combinations?
    • If there is no signal in one of the color channels of the index read, the image registration might fail and no base will be called from that cycle. If no base is called, the index read may not be able to be matched to the sequence specified in the sample sheet, and then samples will not be able to be demultiplexed.

  • Is it okay to multiplex NuPCR assays with different PCR efficiencies? Who should I contact if I need assistance troubleshooting a failed assay?
    • If you are using gene expression assays you can use the PCR efficiencies to normalize them. NuPCR assays are desgiend to have very high PCR efficiencies.
  • Is it possible to connect a bar code scanner to the computer to ease naming of barcoded samples?
    • Currently this is not supported by the Eco system. Check back periodically and keep an eye on the monthly Illuminotes newsletter for updates.

  • Is it possible to generate a larger insert size with TruSeq RNA Sample Prep?
    • See the appropriate TruSeq RNA sample preparation guide for details, Appendix A, Alternate Fragmentation Protocols. Two separate options are provided for varying the insert size of your library:

       

      1. Modify the fragmentation time
      2. Shear the sample after the synthesis of the double-stranded cDNA
  • Is it possible to get the data for each feature on the chip?
    • Yes, this is known as bead-level data. Contact your Field Application Scientist (FAS) or Technical Support Representative for assistance.
  • Is it possible to import standard curves from a previous run?
    • The EcoStudy software, which is available for Eco Real-Time PCR System users, is able to apply a standard curve from one plate across an entire study for data analysis. The software is also able to import an existing standard curve from a previous study for use in analysis.

  • Is it possible to know who submitted the individual samples?
    • Samples are listed as being submitted either by Illumina, or by Other. The User Agreement that is necessary to comply with the Health Insurance Portability and Accountability Act (HIPAA) does not allow the identification of the source of the data from the download tool. However, it is projected that many submitters will reference their study number in their publications.

  • Is it possible to load a custom genome in DesignStudio for Nextera Rapid Capture Custom Enrichment?
    • No it is not.
  • Is it possible to perform QF-PCR on the Eco system?
    • The initial PCR amplification for Quantitative Fluorescence PCR (QF-PCR) can be performed on the Eco system. The subsequent detection and quantification of the amplified fragments requires electrophoresis on a DNA scanner. Some applications of QF-PCR, such as Copy Number Variation (CNV) analysis, can be adapted into Real-Time PCR and be performed on the Eco system.

  • Is miRNA compatible with LIMS?
    • Illumina does not currently offer LIMS support for miRNA.
  • Is performance impacted based on the RNA isolation technique used?
    • Illumina has tested only RNA isolated using RNEasy® from Qiagen®, one of the most frequently used methods. We do not anticipate a major impact on performance with other appropriate, well-established isolation techniques.

  • Is quantitation performed before or after pooling TruSeq Targeted RNA Expression libraries?
    • Samples are quantified after pooling. For more information, see the TruSeq Targeted RNA Expression Guide .
  • Is quantitation performed before or after pooling and gel purification of the TruSeq Small RNA libraries?
    • Samples can be quantified prior to pooling, but this process is often inaccurate.  At high plexity, it may be more effective to pool using equal small volumes prior to gel purification then purify,  amplify and quantitate the pooled library.  Once the pool is sequenced, any samples that did not give sufficient coverage can be re-pooled and sequenced.

  • Is quantitation performed before or after pooling of the libraries during TruSeq DNA PCR-Free Sample Prep?
    • Quantification is performed before pooling the libraries and must be done using qPCR. Other methods will quantify fragments that may not have adapters at both ends and will thus not generate clusters or sequence. Quantification by methods other than qPCR will be inaccurate. It is possible to quantify after pooling if all DNA samples are of similar quality, but this requires very consistent yields and should not be attempted by a new user. See the TruSeq DNA PCR-Free Sample Preparation Guide for details.
  • Is quantitation performed before or after pooling of the libraries during this TruSeq Sample Prep?
    • Samples should be quantified prior to pooling. It is possible to quantify after pooling if all DNA samples are of similar quality, but this requires very consistent yields and should not be attempted by a new user. See the appropriate TruSeq DNA or RNA sample preparation guide for details.
  • Is the ACD1 control required for the TruSight DNA Amplicon assay?
    • The ACD1 control is included to allow customers to assess the success of the library preparation. Illumina recommends that customers who are new to the assay include the ACD1 control. Technical Support will not be able to troubleshoot without this control. If the ACD1 control is included in the sequencing run, it should replace a test sample. For example, if the assay recommends eight samples per run and the ACD1 control is included, then there will be seven test samples plus the ACD1 control.
  • Is the Eco Real-Time PCR system available in block formats other than the 48-well block?
    • Currently, the Eco system is only available in a 48-well format.

  • Is the Eco sample loading dock sold separately?
    • One complete sample loading dock is provided with each Eco system.

       To purchase additional docks:

      1. Go to www.illumina.com/systems/ecoqpcr.ilmn

      2. Click the Options & Accessories tab

      3. Scroll down to the Eco sample loading dock field

      4. Place your order

  • Is the GenomeStudio ChIP-Seq Module compatible with CASAVA 1.8?
    • No.  CASAVA 1.8 generates BAM files which are not compatible with the current version of the GenomeStudio ChIP-Seq Module.  Investigators using the ChIP-Seq Module should run CASAVA 1.7. 

  • Is the GoldenGate Assay for Methylation protocol LIMS-supported?
    • No, there are no current plans to incorporate LIMS support for this product.
  • Is the Infinium Methylation Assay a one-color or two-color assay?
    • This is a two-color assay.

      For the Infinium HumanMethyation27 BeadChip assay, which is based on Infinium I Assay designs, the color incorporated depends upon the base preceding the CpG locus being queried. This can be either green or red.

      The Infinium HumanMethylation450 BeadChip assay includes Infinium I and Infinium II study designs.  In the latter case, a single base extension from the 3' end of the probe sequence (which is one base upstream of the query base) will result in either a red or green signal depending on whether the query site was unmethylated or methylated.

  • Is the TruSeq DNA PCR-Free Sample Prep kit suitable for GC-rich samples?
  • Is the TruSeq Nano DNA Sample Prep Kit compatible with TruSeq Exome Enrichment or Custom Enrichment?
    • The TruSeq Nano DNA kit is not compatible with the TruSeq Exome Enrichment or Custom Enrichment kits. The Nextera Rapid Capture product supports a variety of enrichment applications. For more information, see Nextera Rapid Capture .
  • Is the TruSight One assay compatible with BaseSpace?
    • Yes, BaseSpace can be used for the analysis of TruSight One runs. Illumina recommends using the Illumina Experiment Manager and select Targeted Sequencing and the subsequent Enrichment workflow.  Download VCF files from BaseSpace projects to filter variants using the VariantStudio software.

  • Is the VariantStudio software a clinical software tool?
    • No. The VariantStudio software was developed as a Research Use Only tool.

  • Is the VariantStudio software validated?
    • Illumina VariantStudio has undergone standard software development and testing requirements applied to all Illumina software tools developed for Research Use Only.

      Illumina has not submitted the Illumina VariantStudio software for regulatory approval. The software has not undergone analytical validation or clinical validation required for software tools that are regulated as a medical device. Therefore, it is important that you follow procedures for validation of the software according to your Institution, Local, State, and Federal guidelines.

  • Is the controls dashboard different for the Infinium HumanMethylation27 BeadChip versus the HumanMethylation450 BeadChip?
    • Yes, there are several differences in the control dashboard due to the inclusion of Infinium II Assay designs for the Infinium HumanMethylation450 BeadChip. Please see the Infinium HumanMethylation450 BeadChip User Guide for more details.
  • Is the gel size selection step necessary in the TruSeq ChIP Sample Prep protocol?
    • Illumina recommends performing the gel size selection step as it is designed to remove unligated adapters, as well as any adapters that might have ligated to one another, and selects a narrow 250–300 bp size-range of DNA fragments for ChIP library construction appropriate for cluster generation.

  • Is the incorporation reagent supposed to be a different color?
    • Yes. The incorporation reagent included in the HiSeq v4 SBS kit appears more blue, instead of the purple color of incorporation reagents included in the TruSeq v3 SBS kit.
  • Is the miRNA product a one-color or two-color assay?
    • Illumina's miRNA product is a single-color assay.
  • Is the output of bcl2fastq compatible with CASAVA v1.8.2?
    • Yes, it is compatible. However, bcl2fastq v1.8.4 must be used in place of the configureBcl2fastq step in CASAVA. The output of bcl2fastq v1.8.4 is in the fastq.gz file format organized into project and sample directories as specified in the sample sheet. This output is compatible with the configureAlignment and configureBuild components of CASAVA v1.8.2. The sample sheet format required for bcl2fastq v1.8.4 is equivalent to CASAVA v1.8.2 sample sheet format, and is described in the bcl2fastq v1.8.4 User Guide (part # 15038058) .
  • Is the sample sheet/library sheet optional or mandatory?
    • For MiSeq runs, a sample sheet is required at the start of the run to enable analysis. For HiSeq/HiScanSQ/GA runs, creating and loading this sample sheet at the start of the run is optional, but is highly recommended in order to view data in the new indexing tab of SAV during the run. If you do not load a sample sheet at the start of a run in HCS, you will not be able to view indexing data in SAV. Additionally it is recommended to create the sample sheet in the Illumina Experiment Manager (IEM) prior to performing sample prep in order to confirm appropriate index combinations.
  • Is the version of MiSeq Reporter software used for analysis recorded in the run folder?
    • The MiSeq Reporter software version can be found in the following files located at the root level of the run folder: the log file AnalysisLog.txt, the CompletedJobInfo.xml file, and the workflow-specific results file (e.g. ResequencingRunStatistics.xml).
  • Is there LIMS support for Infinium Methylation BeadChips?
    • LIMS support is available for the Infinium HumanMethyation450 BeadChip.

  • Is there a 300-cycle MiSeq v3 reagent kit?
    • No. Illumina recommends purchasing a 600–cycle kit and discarding the excess reagents after the run. Because of the increased cluster density enabled by v3 runs, this is more economical than running fewer samples with MiSeq v2 reagents.
  • Is there a GenTrain for Infinium Methylation Arrays? Where is my cluster file?
    • Since Infinium Methylation arrays are designed to compare relative methylation levels between two samples or sample groups (such as normal versus tumor, or pancreas cells versus liver cells), there is no GenTrain and/or cluster file for this product. It is similar to doing a paired-sample analysis.

       

  • Is there a PCR-free TruSeq Sample Prep protocol available?
    • The new index adapter design enables PCR-free protocols. (A single cycle of synthesis is required to separate the forked adapter.)  However, for applications that require higher amounts of input for sequencing, Illumina recommends 10 cycles of PCR (with an optional titration for potential cycle reduction and to optimize throughput). The TruSeq DNA LT/HT kits (FC-121-2001, FC-121-2002, FC-121-2003) use 10 cycles of PCR.  The TruSeq DNA PCR-Free LT/HT kits (FC-121-3001, FC-121-3002, FC-121-3003) are optimized for PCR-free applications.  These kits and protocols are not interchangeable.  Please see the TruSeq DNA PCR-Free HT Sample Prep Kit support pages and the TruSeq DNA PCR-Free LT Sample Prep Kit support pages for more information.

  • Is there a change in price with the new MCS4 plus RTE formulation?
    • No, the pricing remains the same.

  • Is there a clear minimum coverage (%) that an alternate allele has to reach to be called a heterozygous (minimum minor allele frequency of 20 or 30%)?
    • The Isaac variant caller is based on a Bayesian model and will assign probabilities to different possible variant calls so no specific minimum exists.
  • Is there a gel-free TruSeq Sample Prep protocol available?
      • TruSeq DNA has a gel-free protocol option (targeted resequencing only).
      • The TruSeq RNA protocols have a gel-free workflow.
  • Is there a limit to the amount of contiguous sequence that can be designed against in TruSeq Custom Amplicon's DesignStudio?
    • There is a 24 kb limit of contiguous sequence that can be entered for a given target region so as not to overwhelm the design server memory resources. For designs spanning very large stretches of DNA we recommend TruSeq Custom Enrichment, a hybridization based enrichment for targeted resequencing.
  • Is there a lower limit of the number of amplicons to use in my pool?
    • To obtain higher quality sequencing data, Illumina recommends sequencing samples with high diversity and avoiding monotemplate stretches during sequencing. Low diversity can occur with pools of one or a few amplicons. Additionally, it is important to maintain color balance for each base of the read or index read being sequenced, otherwise sequencing could fail due to registration failure. Please refer to the MiSeq System User Guide (part # 15027617) for recommendations on low diversity libraries or the Nextera XT User Guide (part # 15031942) for information on low plexity pooling guidelines.
  • Is there a manual way to include or exclude samples from clusters?
    • Yes, in the SNP graph, use the curser to draw a box around the samples you wish to manually edit, right-click and choose the cluster samples should be assigned to, or NC (no call) if you wish to remove samples from any clusters.

  • Is there a maximum distance from the 3' end of the RNA transcript to which probes hybridize?
    • No, there is no set maximum distance. However, strong preference is given to probes closer to the 3' end.
  • Is there a maximum number of rows allowable in the target region File input method in TruSeq Custom Amplicon's DesignStudio?
    • 1000
  • Is there a minimum number of indices I need to use?
    • For dual indexing, at least 2 barcodes/indices need to be present for each Index Read. For less than 6 samples, Illumina recommends using single indexing. Please refer to the pooling guidelines in the Nextera DNA Sample Preparation Guide.
  • Is there a new version of Illumina Experiment Manager (IEM) for use with NextSeq runs?
    • No. For NextSeq, you can record library and indexing information, and specify run parameters from the Prep tab on BaseSpace. When runs are set up on the Prep tab, the run name appears as a selection during the run setup steps on the instrument.

      If you plan to use the bcl2fastq 2.0 to convert base call (BCL) files to FASTQ files on a Linux server, you can optionally use IEM to create a sample sheet. For more information, see the bcl2fastq Conversion User Guide v2.0.

  • Is there a protocol for dual-color hybridization and detection?
    • No. The low variability and high inter- and intra-slide reproducibility of Illumina's arrays make it unnecessary to perform dual-color experiments.
  • Is there a reason why my favorite gene is not included in the TruSeq Targeted RNA Expression predesigned probe list? Will it be added in the future?
    • Every effort was made to include as many transcripts as possible. For human and mouse transcripts, refseq transcript models were used together with the hg19 and mm9 +mm10 genomes, respectively.
  • Is there a rehyb protocol available for HiSeq v4 runs?
    • Yes. Primer rehybridization for HiSeq v4 runs can rehyb the Read 1 primer, the Index 1 Read primer, or the Read 2 primer. Rehyb runs are performed on the HiSeq. For more information, see the HiSeq Primer Rehybridization Reference Guide (part # 15050105) .
  • Is there a separate cost for TruSeq Synthetic Long-Read DNA data analysis?
    • Purchase of the kit includes access to TruSeq Long-Read Assembly or TruSeq Phasing Analysis applications in BaseSpace.
  • Is there a standard panel available for Methylation analysis on the Illumina platform?
    • Yes, our first standard methylation panel is called "GoldenGate Methylation Cancer Panel I". The catalog number for this panel is GM-17-211. It allows the researcher to interrogate 1,505 CpG sites carefully selected from 807 genes where 230 genes contain one CpG site per gene, 462 genes contain two CpG sites and 115 genes have three or more sites. Please refer to the gene annotation text file on our product web page for more information.
  • Is there a tool for applying the adhesive seal to the plate?
    • An applicator tool (squeegee) is included with each Eco sample loading dock.

  • Is there an effect on the data from the MCS4 formulation change?
    • Our goal is to provide as consistent performance as possible and as with any molecular biology technique, however there may be some additional variation. See the Tech Note that describes the results.
  • Is there an optimal cut-off value (i.e., number of tags per peak) to use to eliminate the majority of false positives?
    • See the GenomeStudio ChIP Sequencing Module User Guide , available in iCom and the GenomeStudio Portal, for information.
  • Is there an optimal total number of tags per experiment in order to consider an experiment successful? What coverage is needed to confidently find peaks?
    • Illumina cannot provide an optimal number because it depends on experimental design and antibody specificity. For example, specific transcription factors require a smaller number of tags than those that are more general (PolII, Histone, …). The higher throughput of the GAII system gives confidence that data from one or more lanes in a single flow cell may be sufficient for high confidence in the number of peaks found per region.
  • Is there an option to avoid indels in the TruSeq Custom Amplicon design process?
    • Yes. The ‘Avoid SNPs’ design option will also avoid indels from the given variant database being used (e.g. dbSNP for human designs).
  • Is there any example TruSeq Exome Enrichment beta data that can be shared?
    • Yes, the TruSeq Exome Enrichment Data Sheet contains summary data for a set of exome enrichment experiments. In addition, early access raw data on two DVDs can be ordered from Customer Support or through your local Sales Representative.

  • Is there any reason why I would rather not have stranded information?
    • Projects that are in-progress may keep their current configuration and workflow rather than changing protocols in the middle of the study. Additionally, pooling reads from paired-end stranded runs is equivalent to the same number of reads from non-stranded single read runs (i.e., 2 million reads each of read 1 and read 2 pooled together (4M total) is identical to 4 million reads of read 1 of a non-directional library).

  • Is this product compatible with automation?
    • This process is not currently compatible with automation.
  • Is this product robust against degraded RNA?
    • Yes. Excellent data has been generated using RNA extracted from FFPE tissue samples. Technical reproducibility is highly similar to intact RNA. In addition, Illumina has profiled artificially-degraded RNA samples (95°C heat for 30 minutes). Excellent reproducibility was obtained with these samples. Further, the profiles generated with these samples are comparable to those generated with corresponding intact RNA samples.
  • Is two-round amplification an option?
    • Illumina has not tried any two-round amplification kits. However, some customers have reported successful use of two-round IVT kits with Illumina's gene expression arrays.
  • MiSeq Reporter indicates that I have a sample numbered 0. What is sample 0?
    • Sample 0 is not designated in the samplesheet. Reads that were not successfully assigned to a sample are written to a FASTQ file for sample number 0, and excluded from downstream analysis.
  • Must the Ethnicity field comply to any specific requirements?
    • No, this field is optional, or can be completed with any user-defined categories. Our intent is to provide the most comprehensive and varied dataset, therefore we do not limit ethnicities at this time to certain entries.
  • My OPA tubes arrived at room temperature but the label indicates that they should be stored at -20ºC. Are they still usable?
    • Yes. OPAs are shipped at ambient temperature; stability testing indicates that OPAs are quite stable for short term storage (3-4 weeks) under these conditions. For long-term storage (> 1 month), we recommend storage at -20ºC.

  • My SUD pellets will not resuspend. What can I do?
    • Complete resuspension of the DNA is required for optimal assay performance. Repeat shaking several times to resuspend. If the pellet does not resuspend, store the plate overnight at 4 °C followed by repeat shaking prior to proceeding.
  • My computer has only a CD drive, not a DVD drive. How should I install GenomeStudio software?
    • There are three options:
      --Download GenomeStudio software from iCom.
      --Install GenomeStudio software over a network that has a shared DVD drive or a copy of the GenomeStudio image.
      --Purchase a portable DVD drive with a USB port, then install GenomeStudio software from the DVD.

  • My current system requires periodic optical calibration to ensure performance; how does the Eco system address this?
    • The Eco optical system components do not move during operation, with the exception of the filter slide, which moves while measuring the four emission wavelengths for each sample at each cycle.

      The optical system is calibrated prior to shipment and does not require recalibration. An optional yearly calibration plan is available. If your application requires annual validation, contact Customer Service at 1.800.809.4566 in the U.S. or email orders@illumina.com for pricing and assistance.

  • On how many computers can I install the Eco system software?
    • The Eco system comes with five licenses for the system software, so that you are not constrained to perform the data analysis on the included workstation. You can install the Eco software from the USB drive that is provided with each Eco. If you require more licenses, contact Illumina customer service.

  • Once I place an order, how will I receive my GenomeStudio software?
    • If you have an iCom account and an active system or software warranty, you can download GenomeStudio software from iCom. The installer and associated materials (sample projects, demo data, user guides and release notes) are also distributed on DVD. Customers can place an order for GenomeStudio Module Software & Documentation Sets via their RAM, RSM, or Illumina Technical Support and the order will be filled by Customer Service.
  • One of my NuPCR probe sets is not working. How do I get replacements? Is it possible to choose another design free of charge?
  • Over what range of intensities can I detect the median significant detectable fold change?
    • The intensity range over which a fold change of ~1.35 is significantly distinguishable is greater than 3 logs.
  • Recommendation for FFPE DNA extraction method?
    • We recommend Qiagen QIAamp DNA FFPE Tissue Kit.
  • Should I leave my MiSeq on when not in use?
    • It is best to leave the instrument on at all times. However, if you need to turn off the instrument, perform a maintenance wash first, and then use the Shut Down command on the Manage Instrument screen to safely shut down Windows. If the instrument will not be used within seven days, make sure that you prepare the fluidics system to sit idle. For more information, see the MiSeq System User Guide , Part # 15027617.
  • Should I save *.tif images on my iScan or HiScan for my bead chips?
    • Save *.tif images if you are interested in re-extracting the intensity information with third-party software. While image re-extraction is not supported by Illumina, some users may want to do this for various academic purposes. The 'beadarray' software package can be used and is available at www.bioconductor.org. NOTE: Illumina cannot provide support for this software.
  • Should I use miRNA-enriched samples?
    • Illumina recommends using total-RNA. The enrichment process seems to reduce the precision of the assay, increases the noise due to technical variation, and requires more starting material.
  • Should QC be performed after the in vitro transcription reaction (IVT)?
    • Illumina recommends quantitation of amplified RNA by fluorometry using Molecular Probe's RiboGreen® Reagent. We also recommend additional qualitative analysis with the Agilent Bioanalyzer or by electrophoresis through agarose gel. A less precise RNA quantitation method is the measurement of A260 absorbance with a spectrophotometer.

  • Should TruSeq small RNA libraries be run as single-read or paired-end?
    • RNA libraries should routinely be run as single read (with an index read as desired). TruSeq libraries can be run as paired-end if desired (for example, for directional RNA custom preparations), but this is expected to offer no advantage and significant expense with small RNA libraries.

  • Should the uninterruptable power supply (UPS) for the Eco system be 1.4 kVA, 2.0 kVA, or 2.5 kVA?
    • The Eco system's maximum power draw is 500 VA, which determines the VA rating of the UPS: > 500 VA.

      The average power draw during aggressive Eco cycling is less than 300 VA, which determines the UPS capacity requirement.

      If you want to be able to continue a run during a power failure, then the APC UPS (Part # SUA2200 or SMT2200) is a suitable model, providing approximately 3 hours at 10% of max capacity.

  • The Ambion TotalPrep™ kit is biased for eukaryotic genomes, whereas the microbial mRNAs lack poly-A tails. Does Illumina support any sample prep/labeling kit for microbial genomes?
    • Illumina has not tested kits designed to label microbial RNA. Reagent vendors such as Ambion® do sell kits for this application. These kits may work, but there is no particular kit that we recommend. It is important that a single source of biotin-16-UTP (i.e., same vendor) is used for all labeling reactions (e.g., Ambion #8452 or #8453).

  • The CASAVA calls SNPs at areas where the coverage dips.
    • These SNPs may not be real SNPs, but small indels. A small indel will cause a short run of snp calls (~indel+4) with a concomitant dip in coverage. Check whether the apparent SNP can be explained by a short indel.
  • The Eco system supports standard dyes used for multiplexing; can I use other dyes or new dyes in the future?
    • The Eco optical system supports dyes within four channels, ranging from 505 to 705 nm. The optical system can detect any dyes that fall within those ranges.

      Channel

      Excitation (nm)

      Emission (nm)

      Example Fluorophores Detected

      1

      452–486

      505–545

      SYBR Green I, FAM

      2

      542–582

      604–644

      ROX

      3

      452–486

      562–596

      HEX, VIC

      4

      542–582

      665–705

      Cy5, Q670

  • The TruSeq Nano DNA Sample Prep Kit does not include in-line controls. What positive control can be used instead?
    • Illumina recommends using Coriell Human-1 DNA (NA18507) or Promega Human Genomic DNA (G3041) as a positive control for this protocol.
  • The distribution of fragment sizes seen after tagmentation is much larger than the size distribution of Mate Pair reads seen in my sequencing data. Is this expected?
    • Yes, although the tagmentation reaction may generate a distribution of fragments with an apparent average insert size of 5 kb or more, the smaller fragments present circularize far more efficiently than the larger fragments. Therefore the average fragment size as detected in the sequencing data can be considerably smaller than suggested by the BioAnalyzer electropherogram of the tagmentation reaction.
  • The errorlog.txt file has many entries, such as Unable to copy Queued file (retry later), The network path was not found, or Logon failure: unknown user name or bad password. What should I do?
    • During a typical run, Real Time Analysis (RTA) may occasionally run into copy issues, and will retry copying the file later. When the file is successfully copied, RTA proceeds normally. Therefore, seeing a few of these issues in the error log is acceptable. However, if you see many of these entries (dozens or hundreds), there could be a problem with your network. You might have an incorrect password , network path, or your network connection might not be fast enough. Try the following to troubleshoot this issue:

      1. Make sure that the network location is valid. Your network may be down, or a folder might have been moved.
      2. Make sure that you have the correct user name and password.
      3. Test whether your network is fast enough. See the FAQ “How can I check whether the copying of files proceeds fast enough?” for details.
  • The iControlDB tool requires a Final Case Report (.txt) file. What if I don't have that yet because I am just in the design phase of my experiment?
    • When you downloaded iControlDB, you received template files that can be used in place of a Final Case Report. These template files do not contain sample genotypes, but are in the format required by iControlDB.

  • The installation instructions state that I need Microsoft .NET 3.5 in order to run GenomeStudio software. Where can I get .NET 3.5?
    • You can download .NET 3.5 from Microsoft’s web site.
  • The protocol recommends purifying 200 bp fragments from the gel. What if my fragments are not exactly 200 bps when run on a Bioanalyzer?
    • While 200 bp is the ideal size for the Genome Analyzer, this means the insert will be about 100 bp because the combined flanking sequence (including adaptors) will be about 100 bp. This enables 35 bp to 50 bp single reads. NOTE: If the fragment is less than 150 bp, it might be difficult to obtain 50 bp reads.
  • There are samples from the HapMap Project in the database. What HapMap sample IDs do these Illumina barcodes correspond to?
    •  A list of the Illumina barcodes and their corresponding HapMap IDs is available upon request from techsupport@illumina.com.

  • There is a product listed in iControlDB called “11218540_WGGTManifest.csv”. What product is this?
    • This is the HumanHap550v1 product.

  • There is not a NuPCR distributor for my country. Can I order NuPCR?
    • NuPCR is sold only through distributors. Encourage your regional distributors to contact the Illumina sales team to discuss carrying NuPCR.
  • To what level have you pooled BACs successfully?
    • There are no inherent limits in the software. Illumina scientists have pooled 29 BACs of 130 kb each.
  • To which genome build are the current probe coordinates mapped?
    • The probe coordinates of v1 and v2 MAPS are mapped to Genome build 36.2.
  • Under what conditions and for how long can DNA be stored after bisulfite conversion?
    • Bisulfite-converted DNA can be stored at -20°C for up to one month.
  • Using the IGV, when I zoom in, the Y-axis keeps changing. Can I set it to a fixed value?
    • Yes. Use the Properties tab on the left side to control auto-scaling or to use a fixed scale.
  • What DNA input amount is required for the TruSight DNA Amplicon assay?
    • The assay uses 50 ng of intact, high-quality genomic DNA. DNA extracted from Formalin-Fixed, Paraffin-Embedded (FFPE) tissue is not supported.
  • What DNA input amount is required?
    • The assay uses between 150 and 250ng of DNA, depending on the quality of DNA and number of targets in the CAT.
  • What DNA quantitation method does Illumina recommend for genotyping applications?
    • For genotyping applications, Illumina recommends use of PicoGreen reagent for DNA quantitation. UV-based methods may overestimate DNA concentration by 2–10 fold.

  • What DNA strand is used to design my targets in DesignStudio for Nextera Rapid Capture Custom Enrichment?
    • The forward strand is used.
  • What IEM workflow do I use in for TruSeq Targeted RNA Expression?
    • Select the RNA Resequencing category, then the Targeted RNA application.
  • What Illumina sequencing platforms are compatible with Nextera?
    • Nextera is compatible with all Illumina sequencing platforms: MiSeq , HiSeq 2000, HiSeq 1000, HiScanSQ , and GAIIx.
  • What Illumina sequencing platforms are compatible with TruSeq ChIP libraries?
    • TruSeq ChIP libraries are compatible with all Illumina sequencing platforms, including MiSeq, HiSeq, HiScanSQ and Genome Analyzer.

  • What Illumina software is available for TruSeq ChIP data analysis?
    • CASAVA and (MSR) can be used for demultiplexing. However, they are not intended for TruSeq ChIP analysis.

  • What Nextera Enrichment kits are available?
    • Two different Nextera Enrichment kits are available: an Exome Enrichment kit and a Custom Enrichment kit. Both kits utilize the fast, simple, high throughput workflow of the Nextera technology in combination with a comprehensive enrichment solution. Nextera Exome Enrichment offers 24 indices for enriching up to 48 samples at 12-plex (catalog # FC-121-1204) or 96 samples at 12-plex (catalog # FC-121-1208). Nextera Custom Enrichment includes 14 indexes that create 24 barcode combinations for enriching up to 48 samples at 12-plex (catalog # FC-123-1204) or 96 samples at 12-plex (catalog # FC-123-1208) and 20 indexes that create 96 barcode combinations for enriching up to 288 samples at 12-plex (catalog # FC-123-1224).
  • What Nextera Rapid Capture Enrichment Kits are available and how many samples can be processed with each kit?
    • Kit Name

      Catalog #

      Nextera Rapid Capture Exome Enrichment (8 rxn × 1 plex)

      FC-140-1000

      Nextera Rapid Capture Exome Enrichment (8 rxn × 3 plex)

      FC-140-1083

      Nextera Rapid Capture Exome Enrichment (8 rxn × 6 plex)

      FC-140-1086

      Nextera Rapid Capture Exome Enrichment (8 rxn × 9 plex)

      FC-140-1089

      Nextera Rapid Capture Exome Enrichment (2 rxn × 12 plex)

      FC-140-1001

      Nextera Rapid Capture Exome Enrichment (4 rxn × 12 plex)

      FC-140-1002

      Nextera Rapid Capture Exome Enrichment (8 rxn × 12 plex)

      FC-140-1003

      Nextera Rapid Capture Expanded Exome Enrichment (2 rxn × 12 plex)

      FC-140-1004

      Nextera Rapid Capture Expanded Exome Enrichment (4 rxn × 12 plex)

      FC-140-1005

      Nextera Rapid Capture Expanded Exome Enrichment (8 rxn × 12 plex)

      FC-140-1006

      Nextera Rapid Capture Custom Enrichment (48 Samples)

      FC-140-1007

      Nextera Rapid Capture Custom Enrichment  (96 Samples)

      FC-140-1008

      Nextera Rapid Capture Custom Enrichment (288 Samples)

      FC-140-1009

  • What RNA Amplification kit does Illumina recommend?
    • Two kits have been tested for use with Illumina’s Gene Expression products: Ambion Total Prep Kit (Catalog No. IL1791) and Epicenter TargetAmp-Nano Labeling Kit for Illumina Expression BeadChip(Catalog No. TAN07924). In addition, the Ovation Amplification Kits from NuGen have been shown to produce acceptable results with the use of a modified protocol. Please note that Illumina does not provide technical support for these kits.
  • What TruSeq RNA Access Library Prep Kits are available and how many enrichment reactions can be processed with each kit?
    • The TruSeq RNA Access Library Prep Kit is available in a Set A and a Set B. Each kit contains enough reagents to prepare up to 48 samples. When used together, sets A and B allow for pooling up to 24 samples using the 12 different indexes in each kit. Each kit supports 12 enrichment reactions with 4-plex pooling per enrichment.
  • What TruSight One kits are available and how many samples can be processed with each kit?
    • Catalog Number

      Kit Name

      FC-141-1006

      TruSight One Sequencing Panel (9 samples). Includes MiSeq reagents.

      FC-141-1007

      TruSight One Sequencing Panel (36 samples). Sample Prep only.

      TG-141-1006

      TG TruSight One Sequencing Panel (9 samples). Includes MiSeq reagents.

      TG-141-1007

      TG TruSight One Sequencing Panel (36 samples). Sample prep only.

  • What VCF versions does VariantStudio support?
    • VariantStudio imports, annotates, and analyzes SNPs, insertions, and deletions expressed in VCF versions 4.0 or later. For more information, see VCF version 4.0 on the 1000 Genomes website.
  • What actual assay performance can I expect from my TruSeq Custom Amplicon design?
    • DesignStudio returns high confidence amplicon designs that have delivered unprecedented amplicon multiplexing performance. You can expect to see specificity and uniformity >70%/80% respectively. In practice, we have observed specificity and uniformity >90% for hundreds of designs.
  • What additional equipment do I need with a MiSeq system?
    • The MiSeq includes all the hardware needed for cluster generation, sequencing, and data analysis. More advanced analysis requires additional computing infrastructure. Other equipment may vary with application and sample prep methods, which is outlined in sample prep documentation.

  • What additional equipment is required to run a HiSeq?
    • The HiSeq requires a cBot cluster generation system, a network file system, and a mobile lab bench with locking casters (recommended).
  • What amplicon decontamination protocol does Illumina recommend?
    • Illumina recommends using a combination of UDG and routine lab cleaning with a 10% bleach solution. Also, physical separation between pre- and post-PCR areas is ideal.
  • What applications are suitable for Nextera?
    • Nextera library preparation is suitable for genomic DNA applications where speed is paramount, input material is limiting (e.g., less than 100 ng DNA), where the sample prep throughput is a bottleneck, or when non-mechanical fragmentation is required. For example, Nextera is ideally suited to small genome sequencing with MiSeq.
  • What applications are supported on the HiSeq X?
    • The HiSeq X Ten is intended for large scale human whole-genome sequencing projects using the TruSeq DNA Nano HT Sample Prep Kit, catalog # FC-121-4003. Other applications are not compatible or supported on the HiSeq X.

  • What applications can I run on a MiSeq?
    • The MiSeq system is ideal for amplicon sequencing, targeted resequencing, small genome sequencing, and clone checking. It is capable of performing 16S ribosomal RNA gene sequencing, ChIP-Seq (TF Binding), and small RNA sequencing.

  • What applications have supported workflows on the MiSeq?
    • The MiSeq supports a large portfolio of sequencing applications. See the MiSeq Applications page for more information.

  • What are .idats?
    • An *.idat file is an intensity data file. It contains statistics for every bead type on your BeadChip. The statistics in an *.idat file include the number of beads, the mean, and the standard deviation for each color sample. There is one *.idat file per sample per channel.
  • What are Illumina's recommendations for preparation of ChIP DNA?
    • We do not have any specific recommendations regarding the chromatin immunoprecipitation step, as techniques for this process can vary widely based on the desired application. However, we do not recommend using any type of nucleic acid as a carrier in the chromatin immunoprecipitation prior to sequencing. The carrier nucleic acid is difficult to remove from the sample, and can end up being 50–90% of the final library.

  • What are Illumina’s minimum hardware recommendations to run GenomeStudio software?
    • The table below includes Illumina’s minimum hardware recommendations to run GenomeStudio software.

      Parameter Sequencing Data Analysis Microarray Data Analysis Microarray and Sequencing Data Analysis
      CPU Speed Intel Pentium 2.0 GHz or faster Intel Celeron Duo or faster Intel Celeron Duo or faster
      Processor 64-bit 64-bit 64-bit
      Memory Size 4 GB or more 8 GB or more 8 GB or more
      Hard Drive 250 GB or larger 100 GB or larger 250 GB or larger
      Video Display 1,280 x 1,024 1,280 x 1,024 1,280 x 1,024
      Operating System Windows XP, Vista, or Windows 7 Windows XP, Vista, or Windows 7 Windows XP, Vista, or Windows 7
      Specific OS Requirements Microsoft .NET Framework 3.5 Microsoft .NET Framework 3.5 Microsoft .NET Framework 3.5
      Network Connection 1 GbE or faster 1 GbE or faster 1 GbE or faster
  • What are TruSight DNA Amplicon libraries?
    • TruSight DNA Amplicon libraries are fixed content panels that leverage Illumina’s TruSeq Custom Amplicon (TSCA) assay. TSCA is the fastest and easiest multiplexed amplicon assay optimized for the MiSeq System. It allows for the sequencing of 16–1536 custom amplicons (small regions of interest) across up to 96 samples. The assay also includes on-instrument variant calling for somatic variation with the Somatic Variant Caller on the MiSeq System.
  • What are best practices for reaching the right number of reads per sample?
    • Accurate library quantification is needed to achieve even pooling for enrichment as well as the recommended raw cluster densities. Inaccurate quantitation and pooling can result in higher representation of some samples compared to others in the same pool. Accurate quantification of the final library before loading is needed to reach the recommended raw cluster densities.  Illumina recommends using the same diluted library for both quantitation as well as clustering.  If cluster densities are lower than expected, Illumina recommends checking the library size used in the concentration calculation using a Bioanalyzer trace and adjust accordingly.

  • What are safe stopping points in the Nextera Rapid Capture Enrichment protocol and for how long?
    • There are six safe stopping points in the Nextera Rapid Capture Enrichment protocol:

      1. After First PCR Amplfication at 2° to 8°C for up to 2 days
      2. After First PCR Clean Up at -15° to -25°C for up to 14 days
      3. After First Capture at -15° to -25°C for up to 7 days
      4. After Capture Sample Cleanup at -15° to -25°C for up to 7 days
      5. After Second PCR Amplfication at 2° to 8°C for up to 2 days
      6. After Second PCR Clean Up at -15° to -25°C for up to 7 days
  • What are safe stopping points in the TruSeq RNA Access Library Prep protocol and for how long?
    • There are eight safe stopping points in the TruSeq RNA Access Library Prep protocol:

      1. After Synthesize Second Strand cDNA at -25°C to -15°C for up to 7 days
      2. After Ligate Adapters at -25°C to -15°C for up to 7 days
      3. After First PCR Amplification at -25°C to -15°C for up to 7 days
      4. After Library Validation at -25°C to -15°C for up to 7 days
      5. After First Capture at -25°C to -15°C for up to 7 days
      6. After Capture Sample Clean Up at -25°C to -15°C for up to 7 days
      7. After Second PCR Amplification at 2° to 8°C for up to 2 days
      8. After Second PCR Clean Up at -25°C to -15°C for up to 7 days
      For more information, see the TruSeq RNA Access Library Prep Guide .
  • What are some basic Real-Time PCR terms and their definitions?
    • Some basic Real-Time PCR terms and their definitions are:

      Amplification plot—Plot of fluorescent signal versus cycle number.

      Baseline—The initial cycles of PCR where there is little to no change in fluorescence.

      Threshold—The arbitrary level of fluorescence used for Cq determination. Should be set above the baseline and within the exponential growth phase of the amplification plot.

      Cq (quantification cycle)—The fractional cycle number where fluorescence increases above the threshold. Also referred to as Ct (threshold cycle) or Cp (quantification cycle).

      R—Reporter signal.

      Rn—Normalized reporter signal.

      ΔRn—Baseline subtracted normalized reporter signal.

      Slope—Indicates the efficiency of the reaction. With 10-fold dilutions, a slope of -3.32 indicates a perfect doubling of product per cycle (100% PCR efficiency).

      R2—Reports the linearity of the standard curve.

  • What are some guidelines for improving data quality?
    • With Infinium products, the two main parameters for copy number are the B Allele Frequency (based on genotypes) and the Log R Ratio (based on intensities). The Log R Ratio is the log (base two) of the "observed intensity" divided by the "expected intensity". The "expected" intensity is generated from the cluster file.

      Because of this direct comparison, accurately measuring the sample input amount is vital. Essentially, your input amount should match the recommended value (400ng for Infinium HD Duo; 200ng for Infinium HD Quad and Infinium HD 12-sample products). If this is this case, your Log R Ratio signal will tend to have low noise.

      When the DNA samples are inaccurately quantified, you may see an "undulation" pattern (this looks like waves) in the log R ratio. This tends to be in GC-rich regions of the genome. This wavy pattern makes it difficult to do CNV analysis as the waves themselves look like copy number changes. This tends to confuse algorithms and confound analysis. On the other hand, call rates tend to be only slightly affected by this change (but this varies).
  • What are some third party data analysis tools for TruSeq ChIP samples?
    • There are a number of third party solutions available for the analysis of ChIP data.  These include, but are not limited to, MACS, Avadis, and Partek.  Please note that Illumina cannot provide support for the use of third party software; please contact the software resources directly with any questions regarding the analysis and use of their software.  Additional literature references can be found on the Illumina Epigenetics webpage.

  • What are the DNA input requirements TruSeq Synthetic Long-Read DNA Library Prep?
    • Illumina recommends 50 μl input DNA at 10 ng/μl.

      • Genomic DNA integrity is critical for the success of TruSeq Synthetic Long-Read DNA Library Prep.
      • The DNA must be phenol free, with a size ≥ 40 kb.
      • Illumina recommends a gDNA quality check, using an agarose gel or other instrument, before proceeding with the protocol.

      For more information, see DNA Input Recommendations in the TruSeq Synthetic Long-Read DNA Library Prep Guide.

  • What are the Eco system power requirements?
    • The Eco system runs from a standard wall socket over a wide voltage range (100–240VAC). It does not require 3-phase power. The Eco system ships with a European line cord and a U.S. line cord. The local distributor should provide an appropriate line cord for local power outlet configuration.

  • What are the Eco system's humidity and temperature requirements?
    • Temperature Operating Range         15° to 30° C (59°F to 86° F)

      Storage                                                      10° to 38°C (50°F to 100°F)

      Humidity Operating Range                15–90% RH

      Storage                                                       5–95% RH
  • What are the advantages and disadvantages of TaqMan and SYBR Green I chemistries?
    • The main advantage of TaqMan chemistry is that a fluorescent signal is generated only when there is specific hybridization of the probe to the target sequence. No signal is generated from any non-specific amplification products that were formed during the reaction. Another advantage is that probes can be labeled with different, spectrally distinct reporter dyes, which allows the amplification of multiple target sequences within a single tube (multiplex Real-Time PCR). The main disadvantage of TaqMan chemistry is that design and synthesis of different dual-labeled probes is required for each target sequence, which increases assay setup and cost.

      The main advantage of SYBR Green I chemistry is that it only requires the design and synthesis of two PCR primers, which decreases assay setup and cost. Another advantage for SYBR Green I chemistry is the ability to perform melt curves. The main disadvantage of SYBR Green I chemistry is that since SYBR Green I binds to any dsDNA present during the reaction it will bind to and generate a signal for any non-specific amplification that occurs.

  • What are the advantages of Real-Time PCR over traditional PCR?
    • One advantage of Real-Time PCR over traditional PCR is that it is a closed-tube system requiring no post-PCR processing. Real-Time PCR has higher precision, increased sensitivity (down to one copy), increased dynamic range (greater than 8 logs), and high resolution (less than two-fold differences).

  • What are the advantages of the Gel-Free Protocol?
    • The Nextera Mate Pair Gel-Free protocol is a low input (1 ug), simple, and robust protocol generating Mate Pair libraries with a broad distribution of fragment sizes, from 1.5 kb up to 15 kb with a median insert size of around 3 kb. The Gel-Free Protocol reliably yields high diversity libraries with fewer duplicates, allowing deeper sequencing of the library. Possible applications of gel-free Mate Pair libraries are de novo assembly (especially of smaller genomes) and structural variation detection studies. Although currently not supported by Illumina, the gel-free protocol may also be adapted for automation.
  • What are the advantages of the Gel-Plus Protocol?
    • The Nextera Mate Pair Gel-Plus protocol with size-selection aims to generate mate pair libraries with a narrower size distribution and larger median fragment size. This is achieved by using a gel-based size-selection process to select DNA of a desired size range. A Mate Pair library with a narrow distribution of fragments will facilitate accurate structural variation detection. It will also help de novo assemblies of both simple and complex genomes by providing paired read information which spans larger repeat regions.

      However, the degree of difficulty in generating gel-plus libraries increases as the length of fragments increases. Compared to libraries with smaller Mate Pair fragment sizes, libraries with larger fragment lengths are expected to have a lower final library yield and lower library diversity and are more challenging to make robustly. To increase the robustness of library generation when carrying out the Gel-Plus procedure, several steps in the protocol have been optimized and guidelines are provided.

  • What are the advantages of the Infinium Linkage-12 panel and GoldenGate Linkage V Panels?
    • Infinium Linkage-12 uses the powerful PCR-free Infinium Assay chemistry and protocol at a very attractive price point (the most cost-effective on the market for linkage analysis). GoldenGate Linkage V uses the proven GoldenGate Assay technology and is the linkage product of choice for degraded samples such as FFPE and WGA samples.
  • What are the advantages of the TruSeq Nano DNA Sample Prep Kit?
    • The key advantage to using the TruSeq Nano DNA Sample Prep kit is that only 100 ng of starting genomic DNA is required. This is increasingly important in studies that have limited amounts of DNA or where DNA needs to be split into multiple applications. In addition, the TruSeq Nano DNA Sample Prep kit is a comprehensive solution containing all of the necessary reagents, barcodes, and size selection beads for convenient processing. The improved workflow includes bead-based size selection, therefore eliminating the time of gel-based methods. The kit also produces premier library quality, offering an excellent solution for studies requiring the highest coverage.
  • What are the advantages of using the Decode File Download Utility instead of the CDs shipped with my BeadChips?
    • This software allows you to download an unlimited number of files at one time automatically without physically handling the CDs. This can be done overnight, over a weekend, or one BeadChip at a time, and you can easily select only those files that have not previously been downloaded. You can also select BeadChips based on purchase order (PO) numbers, or you can barcode-scan one or many BeadChips as you receive them. Using this software will likely save you many hours of hands-on time moving and copying files from CDs.
  • What are the applications of HRM?
    • High-Resolution Melt (HRM) curve analysis was originally developed for SNP genotyping but has since been applied to different applications of mutational analysis. Applications for HRM include mutation discovery, DNA fingerprinting, species identification, HLA compatibility typing, allelic prevalence, and DNA methylation analysis.

  • What are the applications of Real-Time PCR?
    • Real-Time PCR has been used in quantification of gene expression, viral quantification, validation of array data, pathogen detection, and allelic discrimination.

  • What are the applications of multiplex Real-Time PCR?
    • Multiplex Real-Time PCR can be applied to relative quantification experiments where the gene of interest and reference gene are co-amplified in the same reaction. Multiplex Real-Time PCR can also be used for allelic discrimination assays, where two differentially labeled probes detect two alleles of a single nucleotide polymorphism. Another application of multiplex Real-Time PCR is pathogen detection, where multiple pathogens can be detected in one reaction.

  • What are the best index combinations for my TruSeq sample prep?
    • Please refer to the TruSeq Sample Preparation Pooling Guide for both single-index and dual-index recommendations and guidelines.

  • What are the best index combinations for my TruSeq sample prep?
    • Please refer to the Pooling Guidelines section in the appropriate TruSeq DNA or RNA sample preparation guide for both single-index and dual-index recommendations and guidelines.

  • What are the call rates for GoldenGate genotyping?
    • The call rates for GoldenGate genotyping for our standard products are generally > 99%. However, for custom OPA design the locus conversion rate is highly dependent on the quality of genotyping assays that are chosen.
  • What are the catalog numbers for TruSeq Rapid reagent kits?
      • TruSeq Rapid SBS kit - HS (50 cycle)—Cat# FC-402-4002
      • TruSeq Rapid SBS kit - HS (200 cycle)—Cat# FC-402-4001
      • TruSeq Rapid PE Cluster Kit—Cat# PE-402-4001
      • TruSeq Rapid SR Cluster Kit—Cat# GD-402-4001
      • TruSeq Rapid Duo cBot Sample Loading Kit—Cat# CT-402-4001
  • What are the computing requirements for the PC Module?
    • The PC Module has the same computing requirements as other GenomeStudio Microarray modules listed here.

  • What are the coverage, read depth requirements, and sample input amounts for sequencing-based methylation applications?
    • Coverage and read depth requirements vary considerably depending on the form of methylation sequencing used. Please refer to the following references for specific recommendations:

      1. Nat Biotechnol. 2010, 28:1026-8.
      2. Nat Biotechnol. 2010, 28:1106-14. Epub 2010 Sep 19.
      3. Nat Biotechnol. 2010, 28:1097-105. Epub 2010 Sep 19.
      4. Nature Reviews Genetics 11:191-203 | doi:10.1038/nrg2732

         

  • What are the differences between the ChIP-Seq sample prep kit and the standard genomic DNA sample prep kit?
    • Sample Preparation Quality DNA that has been chromatin immunoprecipitated with chemical modifications Standard genomic DNA with no modifications
      Starting material 10ng 5μg
      Enzymes / adaptors Less is used More is used
      Product visible on gel No Yes
      Polymerase Standalone Part of pre-mixed component
      Quantitation of final product Requires sensitive method like a BioAnalyzer Less sensitive method required
  • What are the differences between “ChIP grade” and “non-ChIP grade” antibodies?
    • Although we strongly recommend using antibodies that have been shown to produce good results in ChIP-chip experiments, there are exceptions. ChIP grade antibodies were designed specifically for ChIP, but many also work well in other applications and assays. Many antibodies have been successfully tested in ChIP, so please consult directly with the antibody provider for more information. See more info.


  • What are the differences on the GA between version 8.3, 8.4, 8.5 and version 10 recipes?
    • All recipes use latest SBS chemistry but only v10 recipes enable dual indexing and eight-base barcodes for both single-index and dual-index runs.

      • The difference between 8.3 and 8.4 is the introduction of a user wait after Read 1 to load freshly made indexing reagents, which removes the need to load indexing reagents at the beginning of the run to improve index read quality due to aging of reagents.
      • The difference between v8.4 and v8.5 is that the user wait between Read 2 priming and Read 2 resynthesis has been removed. However, a user wait has been added after Read 2 resynthesis and before first base incorporation to load Read 2 SBS reagents.
      • V10 recipes have the same workflow as v8.5 recipes, but are compatible with eight-base barcodes and dual-indexed runs.
  • What are the different starting materials that I can use for Real-Time PCR?
    • The starting materials for Real-Time PCR can be RNA, genomic DNA, or plasmid DNA. RNA must be reverse-transcribed into complementary DNA (cDNA) before PCR. More recently, kits have become available that can perform RT-qPCR (RT = Reverse Transcription) directly from cells without the need for a separate RNA synthesis step.
  • What are the dimensions of the NextSeq 500?
    • For instrument specifications, see the Site Prep/Lab Environment page .
  • What are the effects of putting too much or too little Nextera tagmented library into the enrichment Rapid Capture reaction?
    • Overloading the Rapid Capture reaction with too much library can lead to an increase in off-target reads. Conversely, loading too little library in the Rapid Capture reaction can lead to lower library diversity and an increase in duplicate sequencing reads.
  • What are the effects of putting too much or too little genomic DNA into the Nextera Rapid Capture Enrichment protocol?
    • Overloading the Nextera tagmentation reaction with more than 50 ng of genomic DNA leads to a larger library size distribution which can lower the number of on-target sequencing reads. Less than 50 ng of input DNA leads to a smaller library size distribution and reduced library diversity or elevated duplicate reads, because many of these smaller library sized fragments would be eliminated during the size-selection procedures.
  • What are the effects of putting too much or too little genomic DNA into the TruSight One protocol?
    • Overloading the Nextera tagmentation reaction with more than 50 ng of genomic DNA leads to a larger library size distribution, which can lower the number of on-target sequencing reads. Less than 50 ng of input DNA leads to a smaller library size distribution. The result is reduced library diversity or elevated duplicate reads  because many of these smaller library sized fragments would be eliminated during the size-selection procedures.

  • What are the expected wash volumes following a HiSeq maintenance wash?
    • The HiSeq maintenance wash has three steps: a water wash, followed by a NaOH wash, and then a final water wash. You can expect the following delivered volumes from the eight lines of waste tubing:

      • First water wash-SBS positions only, 32 ml; SBS and PE positions, 72 ml
      • NaOH wash-SBS positions only, 16 ml; SBS and PE positions, 36 ml
      • Final water wash-SBS positions only, 32 ml; SBS and PE positions, 72 ml
  • What are the input DNA requirements for the GoldenGate Genotyping Assay (standard and VeraCode)?
    • 250 ng, 5 µl at 50 ng/µl.
  • What are the input requirements for the Direct Hyb Assay?
    • 50–500 ng of Total RNA, 11 µl at 50–500 ng/µl, according to Ambion Total Prep Kit #IL1791.

      cRNA to load on chips: 750ng in 5 µl (150 ng/µl) for 8- and 12-sample, 1.5 µg in 10 µl (150 ng/µl) for 6-sample.

  • What are the input requirements for the VeraCode GoldenGate Methylation Assay?
    • > 500 ng for two single-use activation reactions, 45* µl at 50–400 ng/µl.

      *According to Zymo Bisulfite Conversion Kit D5001, D5002, D5004. Kit D5003 is not recommended because the elution volume is too large for this assay.

  • What are the input requirements for this kit?
    • For the best results, use 1–10 ug. However, we have successfully used as little as 0.1 µg of UHR Total-RNA when it was highly pure and high quality.
  • What are the insert sizes for TruSeq DNA PCR-Free libraries?
    • The TruSeq DNA PCR-Free Sample Preparation Guide provides guidelines for both a 350 bp and 550 bp average insert size based on paired-end sequencing results. The 350 bp workflow starts with 1 µg of gDNA, and the 550 bp workflow starts with 2 µg of gDNA.
  • What are the key differences between the protocols for genotyping using the GoldenGate Assay and the GoldenGate Assay for Methylation?
    • DNA samples require bisulfite conversion prior to use in the GoldenGate Assay for Methylation but not for genotyping using the GoldenGate Assay. There are four oligos, two allele-specific oligos (ASOs), and two locus-specific oligos (LSOs) required for each assay locus in the Oligo Pool for Methylation Assay (OMA): one probe pair for the methylated state of the CpG site and a corresponding pair for the unmethylated state. The GoldenGate Assay for Methylation is not LIMS-supported. Analysis of methylation data requires the BeadStudio Methylation module.
  • What are the lab temperature requirements for the HiSeq?
    • The lab should maintain a temperature of 19-25°C (22°C ±3°C). This is the operating temperature of the instrument. During a run, do not allow the ambient temperature to vary by more than ±2°C. Maintain a relative non-condensing humidity between 20-80%.
  • What are the lab temperature requirements for the MiSeq?
    • You should maintain a lab temperature of 22°C ±3°C.

  • What are the longest Indels that Isaac has been able to detect reliably?
    • 10bp continuous indel over the length of the read.
  • What are the main components in NuPCR reaction?
    • NuPCR PCR reactions have just 3 components. Dilute NuPCR Master Mix, cDNA target, and NuPCR Assay reagents and you're ready to go.
  • What are the main protocol differences between the Nextera kit and the new Nextera XT kit?
    • The Nextera XT kit introduces a faster tagmentation step and a bead-based library normalization with only 1ng of input DNA. This workflow has been optimized for the Nextera XT kit and reagents between the Nextera kit and the Nextera XT kit are not interchangeable.
  • What are the major detection chemistries used for Real-Time PCR?
    • There are two major detection chemistries used for Real-Time PCR: enzymatic (NuPCR) and hydrolysis (TaqMan) probe-based chemistries, and DNA-binding SYBR Green I dye-based chemistry. Additional detection chemistries include Molecular Beacons, Scorpions probes and LUX primers.

      NuPCR is a probe-based enzymatic chemistry consisting of amplification primers, DNAzyme oligonucleotides, and a universal substrate oligonucleotide. In the reaction sequence, two DNAzyme oligonucleotides (PartZyme A and PartZyme B) bind to the target DNA during annealing to create a NuZyme complex. A universal substrate oligo containing a 5´ fluorophore and a 3´ quencher then attaches to the complex, and PCR amplification primers attach to the DNA, flanking the target sequence on both sides. Detectable fluorescence is produced when the catalytic activity of the NuZyme complex separates the flourosphore and the quencher from each other on the substrate oligo. After the NuZyme complex is released from the target DNA, a copy of the target sequence is created when polymerase binds and extends the target sequence from the PCR amplification primers. NuPCR will continue to repeat the binding and separating process, producing amplification of the DNA and fluorescent signal.

      In TaqMan probe-based chemistry, also known as the fluorogenic 5' nuclease assay, an oligonucleotide probe anneals to a specific sequence downstream of one of the PCR primers. The oligonucleotide is labeled with a fluorescent reporter dye at the 5' end and a quencher dye at the 3'. When the probe is intact, the reporter is in close proximity to the quencher and the fluorescent signal is low as the energy from the reporter will be transferred to the quencher through Fluorescent Resonant Energy Transfer (FRET). During PCR, as Taq DNA polymerase extends from the primers, the 5' exonuclease activity of the enzyme cleaves the annealed probe to separate the reporter dye from the quencher dye, increasing the fluorescent signal.

      SYBR Green I is a dye that binds only to double-stranded DNA (dsDNA) and its fluorescent signal increases only when bound to dsDNA. During PCR, the fluorescent signal of SYBR Green I increases along with the dsDNA amplicon.

  • What are the major differences between the miRNA and DASL protocols?
    • There are four new reagents, Polyadenylation Single (PAS), cDNA Synthesis Single (CSS), miRNA Assay Pool (MAP) and Single-Color Master Mix (SCM). There are also two new steps in the protocol: "Make PAP" for the polyadenylation and "Make CSP" for cDNA synthesis. In addition, the "Make ASE" and "Cycle PCR" steps are modified.
  • What are the minimum controls that I need to run to have confidence in my data?
    • The controls that are required depend on the type of Real-Time PCR experiment:

      • For any Real-Time PCR experiment, use no-template control reactions.
      • If you use reverse transcription, use a no-reverse transcriptase reaction to test for the presence of genomic DNA.
      • For relative quantification, a control (reference) sample as well as a control (reference) gene is required for normalization.
      • In general, use no-template controls to determine if there is contamination. Use non-specific amplification and positive and negative amplification controls to minimize false negative or false positive results.
  • What are the minimum system requirements to run VariantStudio?
    • The minimum system requirements are Windows 7 or later, 64-bit CPU, 2 GB RAM minimum, 4 GB recommended, and 25 MB hard drive space for installation.

      Importing whole genome data or large gVCF files requires more RAM. For more information, see the entry in the File Formats section on this page, "What are the requirements for importing whole genome data or large gVCF files?".

  • What are the network requirements for data transfer from the HiSeq to a server?
  • What are the phases of PCR amplification?
    • There are three phases of PCR amplification: exponential, linear, and plateau. The exponential phase is the first phase of PCR amplification. Reaction components are in excess, there is an exact doubling of product each cycle, and the reaction is specific and precise. Real-Time PCR measures the Cq value at this phase of PCR. The linear phase is the second phase of PCR amplification. The reaction components are being consumed, amplification slows, and the reactions become highly variable. The final phase of PCR amplification is the plateau phase. The reaction is complete and no more products are being generated. Traditional PCR takes its measurements during this phase of PCR.

  • What are the required input amounts for TruSeq Nano DNA Sample Prep?
    • The protocol has been developed and validated with 100 ng of high quality DNA for a 350 bp insert size and 200 ng of high quality DNA for a 550 bp insert size.
  • What are the requirements for importing whole genome data or large gVCF files?
    • Importing large gVCF files requires more memory and takes longer to import. Use the following information as a guideline to estimate required RAM. This information is based on a Quad-Core Xeon® processor with 16 GB RAM.

      • Exome: 500-800 MB RAM per annotated exome
      • Whole genome: 3.5 GB RAM per annotated whole genome, 4 GB RAM recommended in addition to system memory. For example, if you are importing three annotated whole genome files, you need at least 12 GB but 16 GB is recommended to account for system memory.
      • Whole genome gVCF without hom-ref positions: 4.0 GB RAM per annotated whole genome in addition to system memory. VariantStudio imports non-hom-ref positions but it takes longer than 6 hours to go through all 300-400 million lines in the gVCF file. If you are not using the hom-ref import option, Illumina recommends using standard VCF files.
      • Whole genome gVCF with hom-ref positions: VariantStudio populates a maximum of 10 million rows in the Variants table. Therefore, it is not possible to import all positions. When all 10 million lines are populated, > 5 GB RAM without annotation is required. With annotation, 6-7 GB RAM is required. Alternatively, you can pre-process gVCF files for regions of interest before importing to VariantStudio.
  • What are the requirements for the WGS 30x coverage VCF file for TruSeq Synthetic Long-Read DNA phasing analysis input?
    • See the TruSeq Phasing Analysis App User Guide for a list of requirements.
  • What are the sample pooling guidelines for Nextera Enrichment libraries?
    • The Nextera Enrichment DNA Sample Preparation kit supports pre-enrichment pooling of up to 12 different indexed samples. For pooling libraries prior to enrichment, it is recommended to pool libraries so all Index 1 (i7) indexes are unique. With this pooling approach, samples can be sequenced using a single index read workflow, as described in the relevant instrument User Guide. If Index1/i7 indexes are not unique, ensure that libraries with different Index2/i5 indexes are included (e.g. N703/E501 and N703/E502). With this approach, samples can be sequenced using a dual index read workflow, as described in the relevant instrument User Guide. For further details regarding sample pooling, please refer to the “Library Pooling Guidelines” section in the Nextera Enrichment Sample Prep Guide.
  • What are the sizes of data files from the HiSeq?
    • For a dual flow cell 2x101 cycle run (200 Gb) on the HiSeq 2000 using HCS v1.3 and prior, you can expect 2 TB of intensity data (optionally transferred to a server), 250 GB of base call and quality score information, and 1.2 TB of space for alignment output not including 6 TB of disk space used for temporary files removed before completion of alignment. Using HCS v1.4 and Flow Cell v3, storage requirements for raw data are approximately 60% greater than current runs based on additional swath data and increased cluster density.

  • What are the storage requirements using HCS v1.4 and Flow Cell v3?
    • Storage requirements for raw data are approximately 60% greater than current runs based on additional swath data and increased cluster density.

  • What are the system requirements for the Decode File Download Utility?
    • The software will work with any PC that has internet access (port 80) and for which you have rights to install new programs. You may need help from your IT department if you do not have sufficient rights to install new software on your computer. There are no other firewall or security restrictions.
  • What are the workflow changes for analysis/demultiplexing on the MiSeq, HiSeq, and GA for dual-indexed libraries compared to single-indexed libraries?
    • There are no changes for MiSeq analysis. HiSeq and GA data require an upgrade to CASAVA 1.8.2 to demultiplex dual-indexed libraries. It is also recommended to upgrade to SAV 1.8.4 or higher to use the new Index tab for real time demultiplexing information.

  • What are the workflow changes for sequencing on the MiSeq, HiSeq, and GA for TruSeq HT dual-indexed libraries compared to TruSeq v2 libraries?
    • If you are upgrading from an earlier version of software (i.e., HCS 1.3 or lower), please contact Technical Support as you may need to re-validate. Validation of HCS 1.5/RTA 1.13 should not be required if you are upgrading from HCS 1.4/RTA 1.12. The changes in RTA do not affect data quality and the changes in HCS were to update the user interface to enable dual indexing. Please refer to the HCS 1.5 Release Notes for additional information about new features in this software package.

  • What are typical output concentrations from the pre-enriched Nextera Rapid Capture Enrichment library and the fully enriched final library?
    • Typical output concentrations from the pre-enriched library are 40–125 ng/μl.

      Typical output concentrations from the final enriched library are (nM calculation assumes a 400 bp library size):

      • 1-plex: 5–50 nM (1.5–13 ng/μl)
      • 6-plex: 45–130 nM (12–34 ng/μl)
      • 12-plex: 100–250 nM (26–66 ng/μl)
  • What are typical performance metrics of the SNP-Based Linkage IV Panel when my samples are evaluated by Illumina Genotyping Services?
    • Key metrics of Linkage IVb mapping panel: 6000 validated SNP markers, 43% average heterozygosity in Caucasians, 38% in African Americans, 36% Asians, 99.6% reproducibility, 0.006% Mendelian inconsistencies, 99.8% genotype call rate, < 45-day turnaround from receipt of DNA. Performance metrics: Call rate, reproducibility, Mendelian inheritance rate, and heterozygosity are based on studies defined in Illumina's SNP-Based Linkage IV Panel: Design and Validation Technical Bulletin. Performance depends on the quality of the DNA samples.
  • What browsers are supported for MiSeq Reporter?
    • MiSeq Reporter can be viewed with the following web browsers: Chrome 20.0, Firefox 13.0+, IE 9+, and Safari 5.1+.
  • What can I do if my tagmentation reaction has resulted in longer/shorter fragments than I expected? Will this impact my results?
    • The optimal fragment length range for TruSight One libraries is ~300 bp -1 kb. A higher proportion of longer or shorter fragments can affect enrichment and/or clustering. Ensure best practices and Illumina recommended labware and consumables are used.
  • What can cause my Nextera Rapid Capture Enrichment library to look different than the example shown in the Nextera Rapid Capture Enrichment Guide?
    • The quality and quantity of genomic DNA input into the Nextera tagmentation reaction will affect the library size distribution. A larger peak distribution (> 350 bp) can be indicative of > 50 ng genomic DNA input going into the Nextera tagmentation reaction. Conversely, a smaller sample peak distribution (< 225 bp) can be indicative of < 50 ng genomic DNA or fragmented,  low quality genomic DNA.

      It is critical to accurately quantify the concentration of input genomic DNA. Illumina recommends quantifying the starting genomic DNA using a fluorometric-based method specific to double-stranded DNA, such as QuantiFluor or PicoGreen. For more information, see the DNA Input Recommendations section of the Nextera Rapid Capture Enrichment Guide.

  • What can impact the performance of the sample enrichment?
    • This is difficult to predict, but the complexity of the custom oligo library may yield lower enrichment rates and lower coverage uniformity. Additionally, low complexity libraries (e.g. less than 2 Mb capture) or high GC content areas may have lower enrichment or coverage uniformity as well.
  • What causes my NuPCR multiplex design to fail? How do I adjust?
    • There may be interactions between your targets and/or potential assays. Try changing or reversing the dyes-target combinations.
  • What chemistries can I run on the Eco Real-Time PCR system?
    • Eco is an open platform that supports all current Real-Time PCR detection chemistries including DNA binding dyes, hydrolysis probes, as well as other detection chemistries such as molecular beacons and Scorpions primers.
  • What cluster generation and sequencing reagents do I use for MiSeq?
    • The MiSeq System uses the MiSeq Reagent Kit, which includes specially designed and packaged reagents for cluster generation and sequencing.

  • What cluster generation kit should be used for this sample prep library?
    • For kit compatibility information, see the Illumina Version Compatibility Reference.

  • What cluster generation kits are compatible with TruSeq RNA Access libraries?
    • For kit compatibility information, see the Illumina Version Compatibility Reference .
  • What cluster kits are compatible with the Nextera Rapid Capture Enrichment libraries?
    • All paired-end cluster kits are compatible with Nextera Rapid Capture Enrichment libraries. If preparing these libraries for cluster generation for HiSeq, HiScanSQ, or GAIIx, you must use the TruSeq Dual Index Sequencing Primer kit (catalog # PE-121-1003). Use the Read 1 sequencing primer (HP10) in place of the existing read 1 sequencing primer (HP6) in the cBot plate. For more information on using HP10 in the cluster generation process, see the Preparing HP10 for Dual-Indexed Nextera Libraries section of the cBot User Guide .
  • What command line changes are required in CASAVA for processing the dual-index reads?
    • In the demultiplexing workflow (configureBcltoFastq.pl), the --use-bases-mask parameter will need to be included if dual indexing is indicated in the sample sheet: Example: --use-bases-mask=Y*,I*,I* (for single-end runs) or --use-bases-mask=Y*,I*,I*,Y* (for paired-end runs).

      Please see the CASAVA User Guide and release notes for additional user-input commands that are required in CASAVA.

  • What concentration does my RNA need to be?
    • Illumina specifies 40 ng/ul in the assay protocol.
  • What consumables and equipment do I need to process Nextera Rapid Capture Enrichment samples in my lab?
    • A full list of user supplied items can be found in the Consumables and Equipment section of the Nextera Rapid Capture Enrichment Guide .
  • What consumables and equipment do I need to process the TruSeq Synthetic Long-Read DNA libraries in my lab?
    • A full list of user supplied items can be found in the Consumables and Equipment section of the TruSeq Synthetic Long-Read DNA Library Prep Guide or TruSeq Synthetic Long-Read DNA Library Prep consumables and equipment excerpt. Comparable performance is not guaranteed when using alternate equipment.
  • What consumables and equipment do I need to process the TruSeq Targeted RNA Expression samples in my lab?
    • A full list of user supplied items can be found in the Consumables and Equipment section of the TruSeq Targeted RNA Expression Guide . Comparable performance is not guaranteed when using alternate equipment.
  • What controls should I use for sequencing low-diversity samples on a MiSeq?
    • Illumina recommends adding PhiX at 1% to all libraries as an internal control. For low-diversity libraries, the percentage of PhiX depends on the diversity of the library and requires optimization. Monotemplates may require at least 30% PhiX. If you are using RTA 1.17.28 (released with the MiSeq v2.2 updater ) or later, low-diversity libraries requires a minimum of 5% PhiX spike-in. A PhiX control is not required for libraries prepared with the TruSeq Custom Amplicon Kit or the Nextera XT kit.
  • What correlation is expected from technical replicates?
    • At 100- 200 ng total RNA input, R2 = 0.97 is expected.
  • What coverage will I get with TruSeq Targeted RNA Expression?
    • The amount of coverage is dependent on the level of expression of your targets in the samples you are analyzing. TruSeq Targeted RNA Expression assays target specific regions of each transcript and the level of expression depends on whether the region targeted is common between all isoforms of a gene or is transcript specific. Therefore, the amount of coverage obtained for each target is highly variable.
  • What criteria are evaluated in DesignStudio to identify successfully designed NuPCR assays?
    • DesignStudio evaluates many variables including GC content, melting temperature, secondary structure with primers, and specificity against the genome chosen.
  • What criteria determine clusters passing filter on Illumina sequencers?
    • To remove the least reliable data from the analysis results, often derived from overlapping clusters, raw data is filtered to remove any reads that do not meet the overall quality as measured by the Illumina chastity filter. The chastity of a base call is calculated as the ratio of the brightest intensity divided by the sum of the brightest and second brightest intensities.

      Clusters passing filter are represented by PF in analysis reports. Clusters pass filter if no more than one base call in the first 25 cycles has a chastity of < 0.6.

  • What criteria should I use for LOH/CN analysis?
    • You should consider density, physical spacing, and Minor Allele Frequency (MAF). Loci with high MAF (~ 0.5) may be more informative than rare polymorphisms for this type of analysis. The control set must match the test sample population (MAF). You must have control samples with minimal amounts of chromosomal aberrations.
  • What criteria were used for the design of human cSNP assays and what database was used for TruSeq Targeted RNA Expression?
    • Human dbSNP 135 was used for the designs and the following criteria were considered for cSNP assays:
      • cSNP must be common (i.e., those with ≥ 1% MAF have been included)
      • There should be no other cSNP in the probe design area
      • Only 1 SNP per target is allowable
      • cSNP must be covered in all transcripts to a gene
  • What data analysis solutions are available for NextSeq 500?
    • The NextSeq 500 seamlessly streams data to BaseSpace, where demultiplexing and conversion to industry-standard FASTQ file formats occurs automatically as the final step in data transfer to Basespace. BaseSpace provides a number of analysis tools, including the Core BaseSpace Apps for whole genome sequencing, enrichment, and RNA-Seq.

      Illumina provides the bcl2fastq 2.0 conversion software for demultiplexing and conversion of NextSeq 500 output to standard FASTQ file formats. This conversion software enables analysis of NextSeq 500 data in any third-party NGS analysis solutions.

  • What data can I expect from the ChIP-Seq workflow on the MiSeq?
    • The ChIP-seq workflow generates demultiplexed FASTQ files.

  • What data compression options are available for high output runs using HCS v2.2?
    • Two data compression options are zipping of BCL files and binning of Q-scores. Other run folder files are unchanged. These options are available during run setup in HCS v2.2. If you are using BaseSpace for data storage and analysis, BCL files are zipped automatically. Due to the size of the run folder with the extra cycles and shorter run durations, zipped BCL files are required for HiSeq v4 runs. This setting cannot be turned off. You can select or deselect Q-score binning depending on your preference.

  • What differences can I expect if I am analyzing a tumor sample versus a typical congenital sample?
    • Depending upon the number of division cycles, a tumor sample typically has a large number of aberrations across the length of the whole genome. You will see many regions of loss and gain in copy number. cnvPartition will identify many found regions in these samples. Genotyping call rates in these samples may be as low as 60%. The percentage of defects in these samples can be as high as 50% (or even higher). Congenital samples tend to have fewer aberrations and may only have <10 large deletions and duplications. Genotyping call rates in these samples should be above 95% more likely to be 98% or greater. The percentage of defects in these samples should be less than 1%.
  • What do 'Positive Phenotype' and 'Negative Phenotype' mean?
    • These are supplied by the submitter of the data. Samples may have been used as part of the case group in an experimental design (e.g., a sample from a diabetes patient). In this instance, "diabetes" may be reflected in the "Positive Phenotype" for this sample. These samples are still valuable to other studies that are not looking at this Positive Phenotype, and in fact it may be very valuable to obtain accurate population representation in control sets. Similarly,  "Negative Phenotype" is supplied by the submitter and is intended to indicate which phenotypes the sample has been screened for and are negative. Please note that some submitters have chosen also to use the Phenotype field to note the sources of the samples (e.g., blood, lymphocytes). In addition, the entry "HapMap" indicates HapMap samples from Illumina.

  • What do I do if I want to run fewer cycles than 50 or fewer cycles than 300?
    • Use one of the available reagent kits that covers up to the number of cycles you want to perform. Any unused reagents are automatically discarded at the end of the run.
  • What do I do if my AMPure beads are not dry after the 15 minute incubation at room temp?
    • Longer drying times may be required depending on environmental variables and the amount of ethanol remaining in the well.  However, take care not to over-dry beads as this can impact sample recovery.

  • What do I need to do if I want to use another method for relative quantification analysis?
    • The Eco analysis software does not provide alternative methods of relative quantification analysis, but third-party tools are available. You can export all data in *.csv format from the Illumina Eco analysis software and import the data into third-party analysis software such as REST or qBase.

  • What do I need to know to allow VariantStudio access to BaseSpace via my company firewall?
    • VariantStudio needs external connections to annotate. For annotation, VariantStudio simply needs access to the annotation URL via port 80. This URL can be found in the installation folder, in the file VariantStudio.exe.config, next to the key annotationServiceUri. This link should be an Amazon Web Services or Illumina link. In addition to the annotation connection, VariantStudio must make a one-time connection to basespace.illumina.com and icom.illumina.com, also through port 80. This connection serves to make sure that the user has access to the Illumina Annotation Service. This connection is needed again if the Forget BaseSpace Logon option is used, or if the BaseSpace account information is not saved.

  • What do I need to order if I want to run a new Illumina Nextera library?
      • The Illumina Nextera DNA Sample Preparation Kit comes in two sizes: a 24 Sample Kit (Catalog # FC-121-1030) and a 96 Sample Kit (Catalog # FC-121-1031).
      • To complete the sample prep protocol, there are two Index Kits used for PCR: a 24 Index Kit with sufficient reagents for 96 samples (Catalog # FC-121-1011) and a 96 Index Kit with sufficient reagents for 384 samples (Catalog # FC-121-1012). Again, note that each Index Kit is good for four uses and additional caps are included for the tubes to replace after each use in order to prevent cross-contamination.  To assist in correctly arranging index primers during the PCR Amplification steps, a TruSeq Index Plate Fixture Kit is available to order (Catalog # FC-130-1005).
      • When clustering Illumina Nextera libraries on the Cluster Station or cBot and sequencing on a HiSeq 2000/1000, HiScanSQ or GAIIx, new primers are required whether performing a non-indexed, single-indexed, or dual-indexed run. There are two accessory kits available for this: the TruSeq Dual Index Sequencing Primer Kit for Single Read runs (Catalog # FC-121-1003) and the TruSeq Dual Index Sequencing Primer Kit for Paired End runs (Catalog # PE-121-1003). Note that each kit is good for a single run but contains the required primers for both clustering and sequencing.
      • When preparing Illumina Nextera libraries for clustering and sequencing on MiSeq, the appropriate primers are already included in the reagent cartridges: MiSeq 50-cycle Reagent Kit (Catalog # MS-102-1002) or MiSeq 300-cycle Reagent Kit (Catalog # MS-102-1001).
  • What do I need to order?
    • Nextera Enrichment Kits are provided as a bundle with reagents for sample prep, enrichment and the indexes included together under a single catalog part number. To order a Nextera Custom Enrichment kit, DesignStudio is an online design tool that supports custom design and ordering for Nextera Custom Enrichment projects. Please go to http://designstudio.illumina.com/ for more information or to design a project; note that a MyIllumina account is required to access DesignStudio.
  • What do I need to run MiSeq v2 chemistry?
    • Using reagents provided in the MiSeq Reagent Kit v2 requires the recipes and RFID recognition settings in MiSeq Control Software (MCS) v2.0. To download a copy of MCS v2.0, see the MiSeq System downloads page .
  • What do I need to run MiSeq v3 chemistry?
    • Using reagents provided in the MiSeq Reagent Kit v3 requires the recipes and RFID recognition settings in MiSeq Control Software (MCS) v2.3. To download a copy of MCS v2.0, see the MiSeq System downloads page .
  • What do the column headers GeneSymbol, GID, and Accession reference on the gene list for Illumina's standard BeadChips, and where do the numbers come from?
    • Descriptions for all of the column headers can be found in the document "Bead Manifest Field Descriptors" located on the documentation CD included in the startup kit.
  • What do the designators "A", "S," "I," and "M" in the manifest files mean?
    • A = All isoforms. The probe is designed to hit all splice isoforms of a gene.

      I = Isoform specific. The probe is designed to hit a specific splice isoform of a gene, for which multiple isoforms are known to exist.

      S = Single isoform. The gene has only one known splice isoform and our probe hits it.

      M = Multiple isoforms. This gene has multiple isoforms. The probe targets more than one and fewer than all of them.

  • What do the different target Desired Probe Spacing options represent, when designing probes across my Nextera Rapid Capture Custom Enrichment targeted regions?
    • The Desired Probe Spacing setting allows you to adjust the way DesignStudio places enrichment probes within the region of interest. Increasing this setting allows DesignStudio to place probes more closely to one another within a given region. The target center-to-center spacing for neighboring 80 mer probes at each setting is:
      • Standard—230 bp
      • Intermediate—180 bp
      • Dense—120 bp
      • Adjacent—80 bp
      • Overlapping—60 bp
      Note that altering the Desired Probe Spacing setting allows the DesignStudio algorithms to place probes according to the spacing listed, but does not guarantee this spacing. DesignStudio does not place probes that are incompatible with other design rules just to achieve desired spacing.
  • What do the failure codes indicate in my ADT results file?
    • Critical Failures (undesignable):
      • 101 = Flanking sequence is too short.
      • 102 = Polymorphism or sequence formatting error. Possible causes:
        • Check polymorphism format: SNP => [X/Y], INDEL => [-/XYZ], CpG => [CG]
        • More than one set of brackets in sequence
        • Missing brackets around polymorphism
        • SNP alleles not separated by a "/"
        • Spaces found in submitted sequence
      • 103 = Top/Bot strand cannot be determined.
        • Low sequence complexity
      • 104 = Polymorphism is not appropriate for Illumina platform. Possible causes:
        • Tri- or quad-allelic SNP
        • Contains characters other than A, G, C, or T
      • 105 = Polymorphism is located on the mitochondrial genome. Mitochondrial polymorphisms are not recommended for Golden Gate oligo pool.
      • 106 = Degenerate nucleotides in assay design region.
        • W, R, S, N, etc.
      • 107 = SNP sequence not found.
      • 108 = Final score falls below assay limit.
      Warnings:
      • 301 = Polymorphism in duplicated/repetitive region.
      • 302 = Tm outside assay limits.
      • 304 = There are known SNPs within the probe region. See Underlying_SNP column for details.
      • 340 = Another polymorphism in this list is equal to or less than 60 nucleotides away.
      • 360 = Low score warning.
      • 399 = Multiple contributing issues.
      • 601 = Potentially non-specific against the genome.


  • What do the status bar colors indicate on the front of the MiSeq?
    • Green indicates the instrument is ready to run, blue indicates the instrument is running, and orange indicates the instrument needs attention.
  • What do ‘TOP/BOT’ strand and ‘A/B’ alleles mean in the Illumina manifests and reports?
  • What does "design score" mean? What is the relationship between design score and assay conversion?
    • A proprietary algorithm gives a score range from 0 to 1. It is intended as a metric to assist you in selecting SNPs. The higher the score, the more likelihood an assay will convert. This does not guarantee an individual locus with a high score will convert, nor that a low-scoring locus will fail.
  • What does OPA stand for?
    • Oligo Pool All. An OPA contains all of the assay-specific or SNP-specific primers.
  • What does TruSeq Custom Amplicon data look like? Is there demo data available?
    • For an example of data from a TruSeq Custom Amplicon experiment, refer to the data set available via BaseSpace. This example data set is from the TruSeq Amplicon Cancer Panel sequenced with MiSeq on FFPE-derived DNA.
  • What does TruSight DNA Amplicon data look like? Is demo data available?
    • For an example of data from a TruSight DNA Amplicon experiment, refer to the data set available via BaseSpace. This sample data set is from the TruSight Myeloid Sequencing Panel sequenced on the MiSeq with MiSeq v3 reagents.
  • What does amplicon ‘Score’ mean in TruSeq Custom Amplicon's DesignStudio?
    • This score reflects the predicted relative performance of amplicons given Illumina's evaluation of the amplicon probes. It takes into account the target specificity, GC content, and other design parameters.

  • What dyes and specific wavelengths does the Eco system support?
    • The following table details the excitation and emission wavelengths of Eco in each channel. The emission filters combined with spectral de-convolution algorithms effectively minimize cross-talk between dyes. Eco is factory calibrated for SYBR Green I, FAM, HEX, VIC, ROX, Cy5, and Q670. You can use other dyes if they are within the wavelength range of the emission filters.

      Channel Excitation (nm)

      Emission (nm)

      Example Fluorophores Detected

       

      1

      452–486

      505–545

      SYBR Green I, FAM

      2

      542–582

      604–644

      ROX

      3

      452–486

      562–596

      HEX, VIC

      4

      542–582

      665–705

      Cy5, Q670

  • What enrichment levels can I expect when using the TruSight One kit?
    • The expected final percentage of data from enriched regions is 50-70%, but vary across enrichments and can be influenced by the quality of starting material.  The enrichment wash steps are key to reducing non-specific DNA binding and require samples to be maintained at the indicated temperature.  Too low or too high temperatures can result in lower percent enrichments and decreased yields.  Make sure that the proper instrumentation is used and that the streptavidin beads are properly resuspended.  Key points for user-supplied lab equipment, equipment temperatures settings, and enrichment wash procedures are outlined in the TruSight One User Guide .
  • What export formats are available from Illumina VariantStudio?
    • In VariantStudio, you can export information displayed in the Variants table, which includes variants and associated annotations, in a TSV file format. Sample reports are generated in either RTF or PDF file formats.

  • What factors affect the run-time of the BaseSpace TruSeq Long-Read Assembly and TruSeq Phasing Analysis apps?
    • For phasing, the run-time is affected by input short-read volume (GB of short reads). For long-reads assembly, the run-time is affected by GB input, genome complexity, and seeding density.
  • What flow cell should be used with ChIP-Seq libraries?
    • ChIP-Seq libraries constructed with the ChIP-Seq sample preparation kit should be run on single-read flow cells. Libraries constructed with paired-end adapters can be run on either single-read or paired-end flow cells.
  • What flow cell should be used with TruSeq ChIP libraries?
    • TruSeq ChIP libraries can be sequenced using either Single Read or Paired End flow cells.

  • What forms of sequencing-based methylation are there and which do you recommend?
    • There are multiple, published forms of NGS-based methylation, each of which offers its pros and cons. We strongly recommend consulting recent review articles offering comparative analysis of multiple applications. A few examples are given below:

      1. Nat Biotechnol. 2010, 28:1026-8.
      2. Nat Biotechnol. 2010, 28:1106-14. Epub 2010 Sep 19.
      3. Nat Biotechnol. 2010, 28:1097-105. Epub 2010 Sep 19.
      4. Nature Reviews Genetics 11:191-203 | doi:10.1038/nrg2732

         

  • What genes are covered in the TruSight Myeloid assay?
    • The TruSight Myeloid Sequencing Panel targets 54 genes (either full coding sequence or targeted exons) that are frequently mutated in myeloid malignancies. Refer to the TruSight Myeloid Sequencing Panel Data Sheet for a full list of genes.
  • What genomes and databases are used for alignment and variant detection?
    • The MiSeq comes with pre-installed databases, which include miRbase, dbSNP, and refGene. Also included are eight genomes: arabidopsis, cow, DH10b, hg19, mouse, rat, yeast, and s. aureus. You can upload your own references in fasta format (*.fasta or *.fa).
  • What happens if my data connection is interrupted during a NextSeq 500 run?
    • In the event that data transfer is interrupted during a run, data are stored temporarily on the instrument computer until the connection is restored. When the connection is restored, transferring of data resumes automatically.

      If the connection is not restored before the end of the run, data must be removed from the instrument computer manually before a subsequent run can begin.

  • What has Illumina done to ensure a high level of genotyping accuracy?
    • Many factors play roles in determining genotyping accuracy including the quality of the DNA, the quality of the genotyping assay, and the quality of the system. At Illumina we optimize genotyping accuracy by testing samples of DNA before initiating a genotyping project and then by performing quality control procedures on each DNA that we receive. In addition, we have developed a robust genotyping system that is fully automated with built-in internal controls that are run with every genotype. For every SNP assay, our informatics system calls each genotype and assigns a GenCall confidence score. The GenCall score statistically measures the clustering of a particular result against the standard clusters for each genotyping assay.
  • What if I did not name the sample sheet with the reagent cartridge barcode number?
    • During run setup, the software searches for a sample sheet of a name that matches the barcode number of the reagent cartridge scanned in the previous run setup step. If a sample sheet of this name is not found, the software will pause and prompt you to browse to the apppropriate sample sheet for this run, regardless of the sample sheet file name. Naming the sample sheet with the reagent barcode number skips this additional step.
  • What if I don't have enough template DNA?
    • The library process can be optimized to reduce the amount of DNA required. For some applications such as ChIP-Seq where DNA amounts might be low, steps such as size selection on a gel can be skipped. Instead, end-repaired DNA fragments can be ligated, purified by a column clean-up step that removes primer-dimers, and then quantitated and directly introduced to a flow cell for cluster amplification. For genomic DNA from small samples, fragmentation can be optimized to increase the number of DNA fragments that are within the target size range. If these DNA fragments are within a tight size range, then the agarose gel step can be eliminated.
  • What if I have further questions on the MCS4 plus RTE formulation change?
    • Documentation pertaining to this change is available on MyIllumina , particularly the Bulletins section on your MyIllumina home page.  You can also contact Illumina Technical Support .
  • What if I need to run more than 48 samples?
    • The Eco system completes 40 PCR cycles in about 40 minutes, so you can perform multiple runs quickly. The Eco system's superior uniformity provides excellent sample reproducibility and can reduce the number of technical replicates that are required for your experiment. And since it is a personal Real-Time PCR system, you should have access to the instrument virtually any time you need it.

  • What if I want to do an experiment that does not fit into one of the MiSeq Reporter analysis workflows?
    • You can set up your sample sheet to use the GenerateFASTQ workflow, which generates FASTQ files and does not proceed to further analysis. FASTQ files can be exported for analysis by a third-party software.
  • What if Illumina does not use all of the DNA?
    • We destroy or return the remaining DNA to you at any end of the project at your option.
  • What if UCSC doesn’t have a reference genome for the species I am interested in?
    • The genome files for non-human species conform to the UCSC format. The GenomeStudio 2008.1 Framework User Guide , available in iCom and the GenomeStudio Portal, describes how to create genome files for non-UCSC genomes. Illumina is currently working on tools that will convert NCBI formats to UCSC format. Check the GenomeStudio Portal for updates.
  • What if my SNP is not in your database?
    • You can submit the SNP in a SequenceList.
  • What if my hard disk fills up during a download using the Decode File Download Utility?
    • The program automatically checks the disk space available on the directory you select before the download begins, and prompts you if the available space is insufficient. HumanHap300 and HumanHap240S Genotyping BeadChip files are approximately 3.7MB each, and HumanHap550 Genotyping BeadChip files are approximately 6.5MB each. The software checks the available disk space after every *.dmap file, in case another user is filling up the disk space with another process after the initial check (for example, writing BeadScan files to the same disk). The utility alerts you when free disk space approaches 50 MB and stops if the available disk space drops below 50MB.
  • What if the values measured by qPCR and Qubit are very different during TruSeq Synthetic Long-Read DNA library prep?
    • Qubit measurements are substantially higher because it measures total DNA. qPCR measures ligated, amplifiable fragments. It is important to accurately quantify the DNA by qPCR and use this value to calculate dilutions to make sure that the appropriate amount of DNA is used for Long Range PCR.
  • What indexing strategy is used in Nextera Rapid Capture Enrichment libraries?
    • The kits include 12 different Index 1 (i7) adapters (N701–N712) and and up to eight different Index 2 (i5) adapters (E501–E508), depending on the kit you are using. Nextera Rapid Capture Enrichment libraries use the same dual 8 bp indexes as other Nextera based kits. For more information on dual index principles and sample pooling, see the Nextera Rapid Capture Enrichment Guide .
  • What information is captured during my dmap download session?
    • Illumina servers record the login ID and the date, time, and serial numbers of any downloads. This is necessary to allow you to filter your selections during future uses of the software. No cookies or information other than the *.sdf and *.dmap files are written to your computer.
  • What insert size should I use?
    • Between 150 bp and 200 bp is the ideal size range for single-read libraries. The final product of the sample prep process will be about 100 bp longer than the size of the cDNA insert due to the additional length of the Illumina linker and primer regions.
  • What is "Real-Time" Polymerase Chain Reaction (PCR)?
    • Real-Time PCR uses various fluorescent detection chemistries that allow you to monitor the PCR reaction as it progresses. The amount of fluorescent signal generated is directly proportional to the amount of DNA being synthesized during the PCR reaction. Data are collected at each cycle as opposed to traditional PCR, which collects data at the end of the reaction. This allows samples to be characterized by when amplification is first detected as opposed to the amount of product generated after PCR cycling. The greater the amount of the target sequence, the earlier amplification will be detected.

  • What is "information content" and why is it important for a linkage study?
    • Information content is a measure of how informative a marker or map of markers is in a collection of pedigrees in order to extract the maximum amount of inheritance information for a linkage analysis. Information content is a function of marker heterozygosity and the number of meioses in the genetic study. For multi-point linkage analysis, information content is also a function of marker density and spacing. It is important to have high information content throughout the genome for genome-wide searches for disease susceptibility loci or other traits so that regions of no linkage can be excluded, regions of significant linkage can be detected, and the linkage interval can be accurately defined.
  • What is Adapter Trimming?
    • Shorter inserts may lead to sequencing into the adapter, and this feature helps filter out adapter sequence from the final sequence data. Please select this option when creating your sample sheet in IEM for use with MiSeq as the MiSeq Reporter (MSR) analysis software will automatically trim the adapter sequence. For all other run types, this option can be used with the appropriate commands in CASAVA 1.8.2.
  • What is HiSeq X Ten?
    • HiSeq X Ten is a collection of at least 10 ultra-high throughput HiSeq X Sequencing Systems. HiSeq X Ten is capable of sequencing more than 18,000 genomes per year at the unprecendented price point of less than $1000 USD per genome.
  • What is High-Resolution Melt (HRM) curve analysis?
    • HRM is a recent advancement to the traditional melt curve analysis that significantly increases the amount of detail and information that can be captured. It is sensitive enough to be able to differentiate sequence differences within PCR amplicons down to a single nucleotide. Mutations within amplicons are detected as either a shift in the Tm of the product or a change in the shape of the melting curve.

  • What is Illumina's Genotyping capacity?
    • Our genotyping facility was designed as an ultra-high throughput, scaleable service business. Currently, we have capacity to perform well over 1,000,000 genotypes per day.
  • What is Illumina's current regulatory status?
    • For information on Illumina's regulatory, quality, and instrument safety certifications, including ISO certifications, go to the Regulatory and Quality Information page.

  • What is SRF?
    • SRF (Single Read Format) is a generic format for DNA sequence data. The format is defined at http://srf.sf.net.
  • What is TruSeq Targeted RNA Expression?
    • TruSeq Targeted RNA Expression is a sample preparation workflow that enables efficient, multiplexed gene expression profiling for 12–1000 targets per sample and up to 384 samples in a single MiSeq run.
  • What is a CAT?
    • CAT stands for Custom Amplicon oligo Tube, which is the final pool of oligonucleotide probes used to generate amplicons in the assay. A single CAT supports the amplification of up to 1,536 amplicons in a single TSCA reaction.
  • What is a MiSeq manifest folder?
    • The manifest folder contains manifest files required for the Custom Amplicon and PCR Amplicon workflows. A manifest file is required to specify the alignments to a reference and the targeted reference regions used in the workflow. You can specify the location of the manifest folder using the MiSeq software interface. Before you begin the sequencing run, copy the manifest file to the specified location.

  • What is a PhiX spike-in?
    • A PhiX spike-in employs a small amount of PhiX control in the same lane as a sample. This allows real time quality metrics as the PhiX is analyzed during the run. This is not recommended for sequencing a genome with high similarity to the PhiX genome, and does not allow for normalization of data in that lane as per a control lane.
  • What is a TSO?
    • TSO stands for TruSight Oligo, which is the final pool of oligonucleotide probes used to generate amplicons in the assay. A single TSO supports the amplification of all amplicons in a single reaction.
  • What is a TruSeq Targeted RNA Expression add-on project?
    • An add-on project uses a fixed content pool, which can be either a fixed panel or previously ordered custom panel, and allows you to add 12-1000 additional custom targets.
  • What is a TruSeq Targeted RNA Expression fixed content project?
    • Fixed content projects are pre-synthesized oligo pools that you can order. These have been wet-bench tested and designed against various cellular and disease-causing pathways.
  • What is a TruSeq Targeted RNA Expression manifest file?
    • The manifest file is a .txt file which contains the probe and target sequences for your order. It is used by MiSeq Reporter for on-instrument alignment. You must use a manifest file for the analysis of TruSeq Targeted RNA Expression data.
  • What is a beadpool manifest?
    • Also referred to as a SNP Manifest, this is a file containing the SNP-to-beadtype mapping, as well as all SNP annotations. For the Infinium assay, this is a *.bpm file in binary format.
  • What is a cluster file?
    • The cluster file contains the mean (R) and standard deviation (theta) of the cluster positions, in normalized coordinates, for every genotype, for every SNP. The cluster file also includes cluster score information, as well as the allele frequencies from the training set used to generate the cluster file. A cluster file is required for KaryoStudio. Illumina provides a standard cluster file for each product. Alternatively, customers may generate their own cluster file.
  • What is a manifest file and where do I get one for my Nextera Rapid Capture Exome Enrichment project?
    • Manifest files contain a list of genomic regions and coordinates being targeted for enrichment in the Nextera Rapid Capture Exome and Expanded Exome kits. They are also required in the data analysis process for alignment and variant calling in targeted regions. Each product contains a _targeted regions and _probe manifest file.

      Nextera Rapid Capture Exome and Expanded Exome Enrichment manifests can be downloaded from the Nextera Rapid Capture Exome Enrichment Downloads support page.

  • What is a matrix file?
    • The matrix file is used for base calling and accounts for cross talk between dyes.
  • What is a patterned flow cell?
    • A patterned flow cell is a flow cell with billions of ordered nano-wells. Wells have been manufactured into the glass of patterned flow cells allowing for the generation of sequencing clusters in an ordered arrangement. Clusters are aligned more closely together increasing the number of output reads and amount of sequence data generated. The HiSeq X patterned flow cell contains eight lanes and has the same general dimensions as a HiSeq high-output flow cell.

  • What is a tile?
    • A tile is an image captured by the camera on the Genome Analyzer. A flow cell contains eight lanes. Each lane is imaged in two columns with 60 tiles from each column.
  • What is an amplicon and what is the size of an amplicon from TruSight DNA Amplicon libraries?
    • An amplicon is a fragment of DNA that is produced from an amplification event such as PCR. For TruSight Myeloid libraries, amplicon sizes are fixed at a length of 225–275 bp.
  • What is an amplicon and what is the size of an amplicon?
    • An amplicon is a fragment of DNA that is produced from an amplification event such as PCR. For TruSeq Custom Amplicon, amplicon sizes are user-selected when creating a project in DesignStudio and can be 150, 175, 250, or 425 bp in size.
  • What is bcl2fastq 2.0?
    • The bcl2fastq 2.0 software package converts base call (BCL) files generated on the NextSeq 500. If you plan to use a third-party data analysis solution outside of BaseSpace, set the NextSeq to operate in Standalone mode and use the bcl2fastq 2.0 conversion software. For more information, see the bcl2fastq Conversion User Guide v2.0.

      Although bcl2fastq 2.0 is similar to bcl2fastq 1.8.4, version 2.0 is only for use with NextSeq data. Use version 1.8.4 to convert data from HiSeq or MiSeq.

  • What is bcl2fastq?
    • The bcl2fastq v1.8.4 conversion software is a separate piece of standalone software that is run on a Linux scientific computing system. The installer can be downloaded from the Illumina website. System requirements are equivalent to those for CASAVA v1.8.2 and are outlined in the bcl2Fastq User Guide and listed on pages 114–117 of the CASAVA v1.8.2 User Guide (Rev C). If the option to compress BCL files was selected during run setup, then conversion of BCL files requires the use of the bcl2fastq v1.8.4.
  • What is deconvolution?
    • It is the ability to distinguish between two or more clusters that are in close proximity to each other.
  • What is in the tube labeled "Index adapter" in the TruSeq Targeted RNA Index Kit?
    • This tube contains the PCR primers used to amplify the cDNA amplicons. The primer sequences include sequencing primer binding sites and the indices/barcodes.
  • What is included in a Nextera Rapid Capture Enrichment Kit?
    • Nextera Rapid Capture Enrichment kits include all of the reagents needed for generating complete Nextera tagmented and enriched libraries ready for sequencing. The kits also include magnetic SPRI beads for sample clean-up steps. For more information, see the Kit Contents section of the Nextera Rapid Capture Enrichment Guide .
  • What is instrument health data, and where can I find more information about the option to send instrument health data to Illumina?
    • HCS v2.2 allows HiSeq instruments connected to the internet to send instrument health information to Illumina. This information is anonymous and includes only generic run metrics. This information is used by Illumina to help improve Illumina products. If you want to turn off this option or would like further information, see the Options menu in the HiSeq Control Software. You can find the Options menu under Menu, then Tools.
  • What is melt curve analysis?
    • Melt curve analysis is a post-PCR analysis that is compatible with SYBR Green I-based assays. During a melt curve, amplicons produced during PCR are dissociated (or melted) by slowly ramping the instrument from a low temperature to high temperature. The fluorescence is monitored throughout. As the amplification products transition from dsDNA to single-stranded DNA and the SYBR Green I is no longer bound, there is a sharp decrease in fluorescence. The midpoint of this transition is known as the melting temperature (Tm) of the sample and is characteristic of a given DNA sequence. Melt curves are useful for determining the specificity of a PCR reaction, as any non-specific amplification products will have a different melt curve profile than the target sequence.

  • What is multiplex Real-Time PCR?
    • Multiplex Real-Time PCR is a technique in which multiple target sequences are amplified and detected in a single PCR reaction. Amplified sequences are distinguished by the use of different dyes conjugated to the TaqMan probes. The number of targets that can be detected in a single reaction is technically limited only by the availability of spectrally distinct dyes and the ability of the Real-Time PCR instrument to effectively excite and detect those dyes. Some advantages of multiplex Real-Time PCR are reduced reagent costs, reduction in sample use, and increased throughput.

  • What is new about the Nextera Mate Pair Protocol as compared to the Illumina v2 Mate Pair protocol?
    • The new Nextera Mate Pair kit offers several advantages over the previous Mate Pair kit: significantly less DNA input (1-4 ug as compared to 10-20 ug for the original kit), a faster and simplified workflow, and a higher supported read length of 2x250.

      Other advantages of the Nextera Mate Pair kit include utilization of a transposome-mediated fragmentation and adapter tagging of genomic DNA, an identifiable Mate Pair junction sequence, TruSeq DNA Sample Prep master-mixed reagents to reduce reagent containers and pipetting steps, TruSeq DNA Sample Prep adapter indexing compatibility with 12 indexes included in the kit, and a choice of two protocols: a Gel-Free Protocol and a Gel-Plus Protocol.

  • What is paired-end analysis?
    • Paired-end analysis sequences both ends of a DNA fragment. If the fragments are of known size, this method can facilitate de novo sequencing of repetitive elements and help to identify structural variation.
  • What is required in developing multiplex Real-Time PCR assays?
    • Developing multiplex Real-Time PCR assays can be difficult and time-consuming. As the reaction complexity increases, significant optimization may be required to generate reliable data. It can be a challenge to develop multiplex assays that amplify all targets with equal efficiency.

      When developing multiplex Real-Time PCR assays, you need to consider primer design, the relative expression levels of target sequences, and master mix / reagent conditions.

      Use the same design criteria for each primer/probe set and screen all sequences against each other to determine any potential primer-dimer formation. In addition, perform a BLAST analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to determine primer specificity.

      If the expression levels of the target sequences are significantly different, the most abundant target will be preferentially amplified and deplete all the reaction components, compromising amplification of the less abundant targets. One way to address this issue is to limit the primer concentrations of the most abundant target, using the lowest primer concentration that produces the same Cq and PCR efficiency. Limiting the primer allows the most abundant target to amplify and go to completion without depleting all the reagents needed for the other sequences.

      Amplifying multiple target sequences creates additional demand for reaction components. Taq DNA polymerase, Mg++ and dNTP concentrations may need to be optimized to improve amplification of all targets. Master mixes optimized specifically for multiplex Real-Time PCR are now commercially available, and can reduce the amount of time required for optimization.

      Compare experiment data that uses a single assay with the experiment data when the assay is combined into a multiplex assay. Sensitivity and PCR efficiency needs to be the same in both types of assay use.

  • What is required to perform HRM?
    • High-Resolution Melt (HRM) curve analysis requires a different class of dsDNA binding dyes, extremely precise instrumentation, and specialized software.

      HRM analysis is generally performed using dsDNA binding dyes other than SYBR Green I. These dyes are known as saturating dsDNA-binding dyes, and include SYTO 9 (Invitrogen), LCGreen (Idaho Tech), and EvaGreen (Biotium Inc.). These dyes differ from SYBR Green I in that they are significantly less inhibitory to PCR. This reduced inhibition allows them to be used at much higher concentrations than SYBR Green I that saturate the dsDNA amplicons. Greater dye saturation provides greater sensitivity and resolution of melt curve profiles.

      Extremely precise instrumentation is important for HRM. Since some mutations only cause Tm shifts of a fraction of a degree, any thermal or optical non-uniformity will reduce the ability to detect these sequence changes. To be able to perform HRM analysis an instrument needs to have a fast acquisition rate, precise temperature control, and an absolute minimum of thermal and optical variation between samples.

      HRM also requires software with specialized analysis algorithms that can analyze the shape of melt profiles and group similar melt profiles together. HRM data can be viewed as either normalized melt curves or difference plots. Difference plots show the difference in fluorescence from a selected reference sample. Some software also features an auto-call feature, which can automatically assign genotypes based on melt profiles.

  • What is that “clicking” sound coming from the HiSeq or HiScanSQ?
    • You might notice an intermittent clicking sound coming from the right side of the instrument. This is normal. The clicking sound indicates that the refrigeration condensation pump is working. Under normal conditions, the sound occurs for about five minutes in a 12-hour period.

  • What is the DNA input for the Nextera Enrichment kit?
    • For the Nextera Enrichment kits, 50 ng of gDNA is required for input to the prep. Please note that to obtain an accurate quantification of the input DNA, Illumina recommends quantifying the starting DNA library using a fluorometric based method specific for duplex DNA such as the Qubit dsDNA BR Assay system. Upon completion of the prep, 500 ng is used as input for the enrichment portion of the protocol. Illumina recommends quantifying by picogreen or the Qubit Fluorometric Quantitation system. Please note that adding greater than 500 ng of library DNA into the enrichment assay (up to 1 ug per sample) may produce greater mean coverage per sample.
  • What is the Decode File (dmap) Download Utility? How does it work?
    • It is a software application that runs from your PC desktop and enables you to download multiple DMAP (decode) files for Illumina BeadChips more easily than manually copying files from CDs one by one.
  • What is the Diff Score and how does it relate to the p-value?
    • The Diff Score is a transformation of the p-value that provides directionality to the p-value based on the difference between the average signal in the reference group vs. the comparison group. The formula is: DiffScore = 10*sgn(µcond-µref)*log10p; For a p-value of 0.05, DiffScore = ± 13; For a p-value of 0.01, DiffScore = ± 22; For a p-value of 0.001, DiffScore = ± 33 The p-value column is hidden by default. To display this column, use the Column Chooser.

  • What is the HiSeq Analysis Software?
    • HiSeq Analysis Software provides rapid and easy alignment and variant calling for whole human genomes or libraries prepared with the Nextera Rapid Capture exome enrichment kit. For Whole Human Genome Sequencing, HiSeq Analysis Software features the Isaac analysis workflow, which is an ultra-fast and accurate sequence analysis software, providing a 4–6 times speed increase over existing methods. For Nextera Rapid Capture analysis, the BWA alignment and GATK variant calling methods are used. The software can be run through the command line or through a graphical user interface package called Analysis Visual Controller Software (AVC).

  • What is the Infinium OncoArray-500K?
    • The Infinium OncoArray-500K is a custom iSelect BeadChip, designed by the OncoArray consortium to assess cancer predisposition and risk. It is the next generation of the groundbreaking iCOGs array. The OncoArray-500K was designed to allow thorough characterization of the genetic architecture of susceptibility for five common cancers (breast, colorectal, lung, ovarian, and prostate).

  • What is the MCS4 formulation change and reason?
    • Illumina found that separating the enzyme from the buffer provides a minimum of a nine month shelf life from date of manufacture. The MCS3 had a four month shelf life. As a result, the MCS3 has been split into two tubes containing MCS4 (buffer) and RTE (Reverse Transcriptase Enzyme).

  • What is the Polyploidy Clustering (PC) Module?
    • The GenomeStudio Polyploidy Clustering Module (PC Module) can identify clusters for samples where the standard diploid clustering algorithm is inappropriate or not useful, such as for polyploidy organisms like wheat and potato.

  • What is the acquisition time for the Eco system?
    • The acquisition time for all four channels is approximately six seconds.

  • What is the amplicon size in the TruSight Myeloid assay?
    • TruSight Myeloid amplicons are 225–275 bp in length.
  • What is the benefit of increasing the Desired Probe Spacing in targeted regions of my Nextera Rapid Capture Custom Enrichment DesignStudio project?
    • Increasing the Desired Probe Spacing setting (from Standard toward Overlapping) increases the density of probes designed across the submitted target region. For most designs, this means there are more total probes within the region of interest and the likelihood of enriching that region is increased.
  • What is the best way to ensure that my samples have been processed and analyzed without issues?
    • KaryoStudio provides several items for you to examine when you are QCing data. The LogRDev metric provides a measure of noise in the intensity data, and is essentially a measure of standard deviation of Log R values across the autosomes. The "percent aberration" metric is a sum of all of the found regions in a sample divided by the entire length of the genome. In a blood sample, where you expect to have little to no aberrations, you will see a very small (<1%) measure for % aberration. In cases where you have a higher number, it may indicate a sample processing issue. Both of these metrics can be impacted by real biological variation in samples, so they should be examined holistically while taking into account the data viewed in the IGV.

      For specific troubleshooting issues and access to the controls dashboard, load your data into GenomeStudio, if you have access to that software. Otherwise, contact technical support with any additional questions on troubleshooting your data.
  • What is the best way to view the genes in a specific region of interest?
    • Gene information is preloaded into KaryoStudio and displayed in the IGV or in the Genes column of the Found Regions table. In addition, you can use the link to the UCSC Genome Browser, the Database of Genomic Variants, and DECIPHER; all of which provide additional gene information.
  • What is the compelling reason for me to use this methylation tool?
    • The Illumina GoldenGate Assay for Methylation is the first array-based platform capable of high-throughput and multiplexed measurement of DNA methylation status with single CpG resolution. 96 samples per plate with 1,536 separate reactions each = 147,456 quantitative DNA methylation measurements per plate. Flexibility in feature content: oligo sets can be customized and optimized. Entire process complete in less than one week from bisulfite conversion to data analysis.
  • What is the cutover plan for TruSeq DNA and RNA from v1 to v2/LT?
    • TruSeq DNA and RNA v1 kits are no longer available for order. Contact your local Account Manager for assistance in transitioning to the latest TruSeq RNA and DNA Sample Prep Kits for sequencing projects.
  • What is the difference betweeen the TruSeq Nano DNA Sample Prep LS and HS protocols?
    • The TruSeq Nano DNA Sample Preparation Guide contains both a Low Sample (LS) and High Sample (HS) protocol. These protocols differ in the types of plates used and the method of incubation and mixing. The LS protocol uses 0.3 ml PCR plates, the incubation steps are done on a thermal cycler, and mixing method is pipetting. The HS protocol is done in MIDI plates, requires additional equipment such as microheating system for all incubation steps, and mixing method is done on a microplate shaker. The LS protocol is optimized for processing 24 samples at a time and HS protocol for more than 24 samples. Both the LT and HT kits can be used with either the LS or HS protocol and will give comparable results; however, the HS protocol can yield more consistent results between samples.
  • What is the difference between Call Rate and Poly Call Rate in the Samples Table of the PC Module?
    • Call Rate is carried over from the Samples Table in the GenomeStudio Genotyping Module in which the original genotyping project (.bsc) was created. Entries in the Call Rate column do not change when SNPs are clustered in the PC Module. In contrast, the Poly Call Rate is calculated from clustering SNPs in the PC Module and represents the percentage of SNPs for which a given sample was assigned to a cluster.

  • What is the difference between Nextera Enrichment and Nextera Rapid Capture Enrichment?
    • Nextera Rapid Capture Enrichment is an updated, faster enrichment workflow that offers a more efficient exome (37 Mb) in addition to the more comprehensive existing exome product (62 Mb).

      The Nextera Rapid Capture Enrichment kits include similar components as the Nextera Enrichment kits, however formulations have changed for several reagents in the Nextera Rapid Capture Enrichment kits. The enrichment workflow has been shortened by reducing enrichment hybridization time and utilizating more efficient wash steps with an improved washing buffer.

  • What is the difference between TruSeq DNA and RNA Sample Prep v1 and v2/LT Kits?
      • No changes to workflow, increase in index capability
      • Fill volumes and new consumables to support automation
      • Each kit contains 12 of 24 unique indexes and each index reaction sufficient for eight individual samples
  • What is the difference between TruSeq Stranded Total RNA Sample Prep with Ribo-Zero Human/Mouse/Rat and TruSeq Stranded Total RNA Sample Prep with Ribo-Zero Gold?
    • The Ribo-Zero Human/Mouse/Rat kit depletes samples of cytoplasmic ribosomal RNA. The Ribo-Zero Gold kit depletes samples of both cytoplasmic and mitochondrial ribosomal RNA.

  • What is the difference between a lane and a channel?
    • The terms lane and channel are sometimes used synonymously in regards to the eight lanes of a flow cell. However, the term channel may also refer to a color channel on the Genome Analyzer (four colors corresponding to the four bases A, C, G, or T).
  • What is the difference between a relative quantification assay and an absolute quantification assay?
    • A relative quantification (also known as comparative quantification) assay quantifies changes in gene expression relative to a reference gene and reference sample. An absolute quantification assay uses a standard curve of known quantities to determine the quantity of unknown samples. Relative quantification results report fold changes in expression relative to the reference gene and reference sample. Absolute quantification results are reported as an absolute quantity (copies, ng, pg, etc.) extrapolated from the standard curve.

  • What is the difference between one-step and two-step real time RT-qPCR
    • The difference between one-step and two-step real time RT-qPCR lies mainly in the reverse transcription step. In one-step RT-qPCR, a short reverse transcription (5–30 minute) reaction is followed by a PCR reaction in a single tube. In two-step RT-qPCR the reverse transcription reaction takes place in a separate tube. Each method has advantages and disadvantages, depending on the application.

      One-step real time RT-qPCR, which uses gene-specific primers, is useful when analyzing a few genes over a large number of samples. Since both the RT and PCR reactions occur in the same tube, there is less pipetting and sample manipulation, possibly reducing variation and potential contamination. One-step RT-qPCR might not be as sensitive as two-step since its reverse transcription step is much shorter. The reaction conditions needed to support both the RT and PCR reactions might not be optimal for either reaction. Another drawback of one-step RT-qPCR is that it is not possible to archive the cDNA produced during the reverse transcription reaction.

      Two-step real time RT-qPCR is useful when analyzing a large number of genes over a few samples. It supports a flexible priming strategy, allowing for oligo-dT, random primers, or gene-specific primers. Two-step RT-qPCR is generally more sensitive than one-step since the RT reaction is much longer and the RT and PCR reactions occur separately, meaning that they can be optimized individually. Also, the cDNA produced is more stable than the initial RNA sample and can be more easily archived for future use.

  • What is the difference between the Nextera Rapid Capture Exome and Expanded Exome Enrichment?
    • The Nextera Rapid Capture Expanded Exome Enrichment utilizes the same capture oligos as the TruSeq Exome Enrichment Kit paired with the new Nextera Rapid Capture Enrichment reagents.

       

      Exome

      Expanded Exome

      Targeted Region

      ~37 Mb

      ~62 Mb

      Content

      Coding Exons

      Exons, UTR, miRNA

  • What is the difference between the TruSeq DNA PCR-Free LT and HT Sample Prep kits?
    • The TruSeq DNA PCR-Free LT Sample Prep Kits come with adapter index tubes recommended for preparing 24 or less samples at a time. The LT kits come in two sets, A and B, and each set contains 12 unique indices for a total of 24 unique single-index adapters if both kits A and B are combined. Each kit (LT kit A or LT kit B) come with reagents sufficient for 24 samples. The TruSeq DNA PCR-Free HT Sample Prep Kit comes in a 96 well adapter plate format with 96 dual-indexed adapters and enough reagents for 96 samples.
  • What is the difference between the TruSeq DNA PCR-Free sample prep HS and LS protocols?
    • The TruSeq DNA PCR-Free Sample Preparation Guide contains both a Low Sample (LS) and High Sample (HS) protocol. These protocols differ in the types of plates used and the method of incubation and mixing. The LS protocol uses 0.3 ml PCR plates, the incubation steps are done on a thermal cycler, and mixing method is pipetting. The HS protocol is done in MIDI plates, requires additional equipment such as microheating system for all incubation steps, and mixing method is done on a microplate shaker. The LS protocol is optimized for processing 24 samples at a time and HS protocol for more than 24 samples. Both the LT and HT kits can be used with either the LS or HS protocol.
  • What is the difference between the TruSeq DNA and TruSeq DNA PCR-Free protocol?
    • The TruSeq DNA PCR-Free protocol does not have gel size selection or PCR amplification. It utilizes a bead-based size selection in place of the gel. The TruSeq DNA PCR-Free protocol takes about 1 day to complete in comparison to the standard TruSeq DNA protocol which takes 2 days to complete.
  • What is the difference between the TruSeq Synthetic Long-Read DNA library prep long-read and phasing workflows?
    • For the Phasing workflow—

      • 75 fg library is required per well during the Long Range PCR procedures.
      • 15 and 20 PCR cycles are run during the Long Range PCR procedures.
      • BaseSpace TruSeq Phasing is used for analysis.

      For the Long-Read workflow—

      • 3 fg library is required per well during the Long Range PCR procedures.
      • 21 and 26 PCR cycles are run during the Long Range PCR procedures.
      • BaseSpace TruSeq Long-Read Assembly is used for analysis.
  • What is the difference between the TruSeq sample prep HS and LS protocols?
    • The TruSeq sample prep high sample (HS) protocol requires additional peripheral equipment, but reduces user touch-points and is ideally suited for projects that include more than 48 samples prepared at one time. The low sample (LS) protocol requires minimal peripheral equipment, but requires more user manipulation and is best suited for projects that include 48 or fewer samples. For specific differences in the workflows, including required equipment, refer to the user guide for the TruSeq sample prep kit that you are using.

  • What is the difference between the TruSeq sample prep HT and LT kits?
    • TruSeq high throughput (HT) sample prep kits each include reagents for 96 samples divided equally between two tubes. Each kit also includes an adapter plate containing 96 unique dual-indexes.

      The TruSeq low throughput (LT) sample prep kits (A and B) each include reagents for 48 samples and 12 of 24 total adapters.

      The length of the HT and LT adapters differ. Please see MyIllumina Support Bulletins  for the sequences.

      There is only one minor protocol change in that the amount of AMPure XP beads has been adjusted for the PCR clean-up for the HT kit. This has been optimized for the longer adapters present in the adapter plate. Please see each kit's user guide for more information.

  • What is the difference between the TruSight One and Nextera Rapid Capture (NRC) Exome and NRC Expanded Exome products?
    • TruSight One is a focused sequencing panel that enriches sample libraries for a selection of coding exons associated with human disease, capturing ~12 Mb of genomic content. The NRC Exome kit targets >98% of coding human coding exon content and captures ~37 Mb of genomic content.  The NRC Expanded Exome panel includes NRC exome content and additional UTR, promoter, and miRNA targets with ~62 Mb  genomic content.  TruSight One and the Nextera Rapid Capture Exome and Expanded Exome kits all use Illumina Rapid Capture library prep technology.

  • What is the difference between the open source version of the Isaac algorithms and the HiSeq Analysis Software? Which one should I use?
    • The Open Source version of Isaac includes the component algorithms for the Isaac aligner and Variant Caller and is intended for developers. This version is not commercially supported and is provided as is under Illumina Open Source Software License available here.

      For the majority of Illumina customers,  we recommend using Isaac as provided as part of the HiSeq Analysis Software package.

      HiSeq Analysis Software provides rapid and easy alignment and variant calling for Whole Human Genomes (using the Isaac component algorithms) or libraries prepared with the Nextera Rapid Capture (NRC) exome enrichment kit (using the BWA/GATK component algorithms). The software can be run through the command line or through graphical user interface package called Analysis Visual Controller Software (AVC). HAS is freely available, easy to install (rpm) and commercially supported. For more information, see our Support page. http://support.illumina.com/sequencing/sequencing_software/hiseq-analysis-software.ilmn

  • What is the difference between the v1.5 small RNA and TruSeq small RNA sample preparation kits?
      1. The TruSeq kits allow indexing of up to 48 samples per lane. The v1.5 kits do not support indexing.
      2. The TruSeq kits have been optimized to simplify the workflow and reduce adapter dimer formation.
      3. The TruSeq kits allow paired-end sequencing, useful for directional RNA sequencing.
  • What is the difference difference between the TruSeq Nano DNA LT and HT Sample Prep kits?
    • The TruSeq Nano DNA LT Sample Prep kits come with single-index adapter tubes recommended for preparing 24 or less samples at a time. The LT kits come in two sets, A and B, and each set contains 12 unique single-index adapter tubes for a total of 24 unique single-index adapters if both kits A and B are combined. Each kit (LT kit A or LT kit B) come with reagents sufficient for 24 samples. The TruSeq Nano DNA HT Sample Prep Kit comes in a 96 well adapter plate format with 96 dual-indexed adapters and enough reagents for 96 samples.
  • What is the difference in design between the Cancer Panel and a custom TSCA panel?
    • The cancer panel is offered as an off-the-shelf standard, or fixed panel that cannot be customized. TruSeq Custom Amplicon offers the ability for users to fully customize targets of interest using DesignStudio.
  • What is the different between Nextera Rapid Capture Exome Enrichment and TruSight Exome?
    • The Nextera Rapid Capture Enrichment Exome is designed to cover >98% of coding content of the human genome, spanning ~37 Mb of genomic content. The TruSight Exome is a focused subset of the exome targeting 7 Mb of genomic targets associated with a variety of inherited diseases.
  • What is the dynamic range of NuPCR assays?
    • The range is up to 7 logs. In practice most experiments are limited by the biological sample or the qPCR instrument.
  • What is the energy consumption of the Eco system?
    • The peak power of the instrument is 500 VA. Typical power consumption during a run is around 180 VA.

  • What is the error rate on MiSeq?
    • Based on the latest advances in Illumina's reversible terminator SBS chemistry, the MiSeq System is the most accurate sequencing instrument available, providing the world's highest output of perfect, error-free reads. For examples of MiSeq performance, see the MiSeq publications page.

  • What is the estimated genomic pull down region from a probe designed in DesignStudio for Nextera Rapid Capture Custom Enrichment?
    • The estimated region is 280 bp total with the probe at the center.
  • What is the estimated overall time for preparing libraries with the Nextera XT kit?
    • The estimated time for 8 samples is approximately 90 minutes for the prep and 70 minutes for the bead based normalization (BBN). The estimated time for 96 samples is approximately 3 hours for the prep and 90 minutes for the BBN.
  • What is the estimated success for sequencing custom targets with the Nextera Rapid Capture Custom Enrichment workflow?
    • While coverage depth of individual target regions may vary, all targeted regions should be represented in the final enriched library.

      A very low percentage of targets may not be represented in the final enriched library due to complex genomic regions, low probe specificity, skewed GC content, and other technical factors inherent to probe based enrichment and SBS. DesignStudio includes Design Warnings to highlight probes with an elevated risk of underperformance.

  • What is the expected correlation for hybridization replicates between BeadChips? Between arrays within a BeadChip?
    • r2 between BeadChips: .99

      r2 within BeadChip: .99

  • What is the expected percentage of remaining rRNA after TruSeq Stranded Total RNA Ribo-Zero treatment?
    • For standard (non-FFPE) samples less than 6% of ribosomal RNA contamination is expected. For FFPE samples, less than 6% is expected. These values vary depending on tissue quality and source.
  • What is the expected size of a MiSeq analysis folder?
    • The size of the analysis output folder for each sequencing run depends on the number of cycles in your run. Typically, a 150-cycle paired-end run (2 x 151 cycles) generates approximately 3 GB in output.

  • What is the general workflow for designing a Nextera Custom Enrichment project in DesignStudio?
      • Choose a project name and details (genome builds, etc)
      • Submit targets with source and build
      • Choose exons or full regions
      • Design is automatically executed
      • Visualize probes
      • Choose probes to place in final design
      • Review design
      • Place order
  • What is the input amount for each protocol and how many samples can I prepare using the Nextera Mate Pair kit?
    • When following the Gel-Free protocol, the recommended input is 1 μg of genomic DNA and the kit is formulated to generate 48 libraries from 48 independent samples. When following the Gel-Plus protocol, the recommended input is 4 μg of genomic DNA and this kit is formulated to either prepare 12 libraries with single size-selections per sample, or up to 48 libraries if making as many as 4 size-selections from each 12 inputs.
  • What is the input amount required for TruSeq DNA PCR-Free Sample Prep?
    • 1 µg of high quality gDNA is required for final libraries with an average 350 bp insert size and 2 µg of high quality gDNA is required for a 550 bp insert size.
  • What is the input amount required for TruSeq Sample Prep?
      • ChIP: 5–10 ng ChIP-enriched, fragmented DNA
      • DNA: 1 μg gDNA
      • DNA PCR-Free:
        • 1 µg gDNA for a 350 bp insert size
        • 2 µg gDNA for a 550 bp insert size
      • Nano DNA:
        • 100 ng gDNA for a 350 bp insert size
        • 200 ng gDNA for a 550 bp insert size
      • RNA: 0.1-4 μg total RNA
      • Small RNA: 1 μg total RNA
      • Targeted RNA Expression:
        • 50 ng total RNA
        • 100-200 ng degraded or FFPE RNA, depending on fragment size
  • What is the intensity spike after template generation?
    • For HiSeq v4 runs, there might be a noticeable increase in the reported intensity by cycle in SAV after template generation has completed after cycle 5. This increase occurs at this point because the reported intensities include only the clusters included in the final template. Before template generation, total intensity is reported.
  • What is the length of the oligo baits used in Nextera Rapid Capture Enrichment reactions?
    • The coding Exome (37 Mb) and Custom content assays use 80 mer oligos, while the Expanded Exome (62 Mb) uses 95 mer oligos.
  • What is the length of the oligo probes used in Nextera Rapid Capture Custom Enrichment reactions?
    • The read length is 80 bases.
  • What is the library loading concentration required for sequencing on the NextSeq 500?
    • To reach a desired cluster density dilute denatured libraries to a loading concentration of 3 pM for most library types. The appropriate loading concentration can vary depending on library preparation and quantification methods.
  • What is the library size distribution for Nextera libraries?
    • The Illumina Nextera DNA Sample Preparation kit produces libraries with a broad size range distribution, typically between 300-1000bp. Please refer to the Nextera DNA Sample Preparation Guide for more specific details.
  • What is the library size distribution for TruSeq ChIP Sample Prep libraries?
    • The final library should show a size distribution from ~250–300 bp.

  • What is the limit of detection for the miRNA assay?
    • The limit of detection is 100,000 molecules when spiked in the background of 200 ng total RNA.
  • What is the liquid inside the Eco system thermal box? Is it dangerous or toxic?
    • The fluid contained in the heating block is specifically designed for use in heat transfer applications and has been used for the last 30 years as an electronic coolant. The fluid is hermetically sealed in each block, is environmentally benign, and has a low vapor pressure.

  • What is the lowest detectable fold change?
    • During product development experiments, the lowest detectable statistically significant fold change was 1.2 fold.
  • What is the major difference between the DBSCAN and OPTICS algorithm, and how do I choose between them?
    • OPTICS is an acronym for "Ordering Points to Identify Clustering Structure". It is a sub-algorithm of DBSCAN, developed to be more robust to changes in input parameters. This trait makes OPTICS more suited for initial clustering. DBSCAN is an acronym for "Density-based Spatial Clustering of Applications with Noise". This algorithm is more sensitive to initial input parameters such as cluster distance. DBSCAN is more suited for differentiating clusters that are very close together, and should typically applied to SNPs for which OPTICS does not yield satisfactory results. 

  • What is the maximum amount of amplicons I can sequence on a MiSeq?
    • If you are using the TruSeq Custom Amplicon Kit, 96 samples can be pooled in a single run combining up to 384 amplicons per sample.

  • What is the maximum amount of input RNA that I can use for TruSeq Targeted RNA Expression?
    • Illumina has tested up to 500 ng of high quality RNA in starting material. However, no improvement in yield is seen in the final amount of RNA generated beyond 200 ng of starting material for high quality RNA.
  • What is the maximum number of reads, lanes, or flow cells that can be loaded into GenomeStudio software?
    • As of January 2009, approximately two billion rows can be loaded, and 128 million rows can be displayed in the Sequence Table.
  • What is the maximum sized genome I can sequence on a MiSeq?
    • For best results, Illumina recommends small genome (up to 20 Mb) sequencing on the MiSeq.

  • What is the median statistically significant, detectable fold change of the GEX BeadChips?
    • 1.35 fold
  • What is the minimum PhiX amount that can be spiked in?
    • For most libraries, Illumina recommends using a low-concentration spike-in (1%) of PhiX Control v3 as an in-lane positive control for alignment calculations and quantification efficiency.
  • What is the minimum aberration size I should use to filter my data?
    • This varies for each user depending on the type of cytogenetics lab in which the samples are analyzed. For example, some cytogenetics labs tend to look at all aberrations greater than 250kb in size. Others may look at aberrations 100kb or greater in size. Therefore, it is up to the user to decide.

      You may want to set a threshold for the minimum number of SNPs per region, such as 50. This information can be found in the # SNPs column in the Found Regions table.

      Remember that more aberrations appear in the Found Regions table when you allow for regions of a smaller size. In this case, many of the regions may not yet be linked to a specific phenotype. Therefore, users may want to try a "top-down" approach, beginning with the largest regions that may be most likely already linked to a phenotype. Depending on the product in use, you may find aberrations as small as 1kb in size.
  • What is the minimum amount of samples I have to prepare through the Nextera Rapid Capture Enrichment protocol?
    • One sample can be prepared at a time. However:
      • When using the Nextera Rapid Capture Exome Enrichment 8 rxn kits, it is most economical to process and pool 8 samples. However, pooling is not performed when using the 8 rxn × 1 plex kit.
      • When using the 12 plex or 288 sample kits, it is most economical to process and pool 12 samples through Nextera Rapid Capture Enrichment for compatibility with the reagent kit configuration.
  • What is the minimum amount of samples I have to prepare through the TruSeq RNA Access Library Prep protocol at one time?
    • One sample can be prepared at a time, however you will not have enough reagents to process all 48 samples. The kit is designed for 4-plex pre-enrichment pooling.
  • What is the minimum amount of total RNA I can use with the Ambion TotalPrep™ Kit?
    • Illumina recommends a minimum of 50 ng total RNA. This generally produces 5-10 ug of amplified RNA, which should be sufficient for 10-20 arrays.

  • What is the minimum data required and maximum sequencing data used for TruSeq Synthetic Long-Read DNA phasing analysis and long-read assembly?
    • A minimum of 30 GB of short-read data is recommended. A warning is given for data sets < 30 GB. Analysis of < 30 GB data may result in poor phasing or assembly results. The maximum input is 115 GB of short read data. A warning is issued if this limit is exceeded.
  • What is the minimum gap size required between adjacent target regions in TruSeq Custom Amplicon's DesignStudio?
    • A gap equivalent to an amplicon size is required between target regions; regions with less than this gap size are merged to improve design performance and prevent unfavorable probe to probe interactions.
  • What is the minimum input material for small RNA sequencing?
    • The protocol was optimized using 1.0 μg of total RNA.
  • What is the minimum number of samples that I can multiplex with my Nextera Rapid Capture Custom Enrichment probe pools?
    • Post-enrichment library yields may be insufficient for clustering and sequencing if doing low-levels of multiplexing of small capture target panels. Illumina recommends avoiding low levels of sample multiplexing (e.g., < 6-plex) for a small capture target size (e.g., < 2 Mb). For large custom designs multiplexing down to 1x can be achieved.
  • What is the minimum order I can place?
    • The minimum order is 48 samples (which is the smallest kit size).
  • What is the minimum recommend total-RNA starting amount?
    • Illumina recommends starting with 100 ng of total-RNA. However, highly reproducible (R2 0.94) miRNA expression profiles have been generated with as little as 2 ng total RNA input.
  • What is the necessary length of input material (i.e., if starting from DNA that is already fragmented, amplicons, etc)?
    • Illumina recommends >300bp to ensure even coverage across the length of the DNA fragment. An expected drop off in sequencing coverage about 50bp from each distal end of a fragment may be seen. This is because the tagmentation reaction cannot add an adapter right at the distal end of a fragment. For PCR amplicon sequencing this can be easily averted by simply designing your amplicons to be ~100 bases larger than the desired insert to be sequenced.
  • What is the noise level of the Eco system?
    • The Eco system is extremely quiet, producing less than 62 db during a run.

  • What is the optimal cluster density on the HiSeq?
    • The recommended maximum cluster density is 750,000–850,000 clusters/mm² when using Illumina's v3 cluster generation and sequencing reagents in combination with HCS v1.4.
  • What is the output file format from a run on the NextSeq 500?
    • The NextSeq 500 generates base call (BCL) files aggregated by lane with one BCL file for each lane, for each cycle. The aggregated file contains the base call and associated quality score for every cluster for that lane. For each lane, one base call index file lists the original tile information in a pair of values for each tile.

      When using BaseSpace, BCL files are automatically converted to FASTQ files when data transfer is complete. If BCL files are saved to a local server, use bcl2fastq 2.0 to convert base calls from a NextSeq run. This FASTQ converter must be run on a Linux server.

  • What is the output of MiSeq?
    • The output is greater than 7 Gb (2 x 250 bp read length) and greater than 15 million reads/tags (based on cluster density). For more information, see the MiSeq System Product Information Sheet.

  • What is the overlap between SNPs on the Infinium OncoArray-500K and other Illumina arrays?
    • The arrays listed below have the following overlapping SNPs:

      HumanCore v1

      246,557 SNPs

      HumanCoreExome v1-1

      260,852 SNPs

      OmniExpress v1-1

      275,691 SNPs

      iCOGs

      40,569   SNPs

  • What is the overlap in targeted genomic content between the Nextera Rapid Capture Exome and Expanded Exome Enrichment products?
    • 33.0 of the 37.1 mega bases targeted in the Nextera Rapid Capture Exome Enrichment overlap with the 62.1 mega bases of regions targeted in the Expanded Exome Enrichment pool. BED files outlining both the overlapping regions as well as the content unique to each product can be downloaded from the Nextera Rapid Capture Exome and Expanded Exome Enrichment Kits Downloads support page.
  • What is the plan for legacy Illumina sample prep kits?
      • mRNA-Seq Sample Prep Kits - available until the end of 2011.
      • Genomic and Paired-End Sample Preparation Kits are currently available for order. Customers will be notified of any changes to the availability of these kits.
      • Small RNA Sample Preparation Kits - available until the end of 2011.
      • Genomic, Paired-End, Multiplexing Oligo Only Kits are currently available for order. Customers will be notified of any changes to the availability of these kits.
  • What is the read length on MiSeq?
    • You can perform up to 2 x 250 bp, or 500 cycles of sequencing on the MiSeq using the MiSeq Reagent Kit v2. However, as few as 36 cycles can be used for some applications.

      Using the MiSeq Reagent Kit v3, you can perform up to 2 x 300 bp, or 600 cycles of sequencing. MiSeq Reagent Kit v3 is available in two sizes, 600 cycles and 150 cycles.

  • What is the reference database for ADT: i.e., the source and version for sequence, Minor Allele Frequency (MAF), and validation?
    • ADT retrieves data based on dbSNP126 for sequence, position, and MAF.
  • What is the reproducibility of the GoldenGate for Methylation Assay?
    • The average reproducibility between technical replicates is > 0.98.
  • What is the sensitivity for Illumina Gene Expression BeadChips?
    • =1 in 250,000
  • What is the sequence of the Mate Pair junction adapter?
  • What is the shelf life for NuPCR reagents?
    • The shelflife of NuPCR Assays and Master Mix is 12 months from the date of manufacture when stored at -15° to -25°C .
  • What is the shelf life of the MiSeq Reagent Kit?
    • Illumina guarantees that all reagent products will ship with a minimum viable shelf-life of three months.
  • What is the shelf life of the TruSeq HT sample prep kits?
    • One year from the date of manufacture. The kit will contain an expiration date on the label.  Illumina guarantees at least 3 months from the date of receipt. This is the same as the TruSeq DNA LT and TruSeq RNA v2 kits. 

  • What is the shelf life of the TruSight One Sample Prep kits?
    • Expiration dates are printed on the kit box labels and reagent tubes. Illumina guarantees 3 months of shelf life from the time of kit shipment. 

  • What is the shelf-life of TruSeq Synthetic Long-Read DNA library prep kits?
    • Expiration dates are printed on the kit box labels and reagent tubes. Illumina guarantees 3 months of shelf life from the time of kit shipment.
  • What is the size of the TruSeq Exome Enrichment capture probes?
    • ~95 bases
  • What is the smallest batch size that can be prepared with the TruSeq Amplicon - Cancer panel kit?
    • As with TruSeq Custom Amplicon, the smallest supported batch size is 16 samples (15 samples plus one control). The 16 sample minimum batch size accounts for up to 6 freeze/thaw events of the 96 sample kit.
  • What is the storage capacity of the MiSeq integrated computer?
    • The integrated instrument computer has approximately 550 GB of storage capacity. For greater storage capacity, you can securely analyze, store, and share your MiSeq data using BaseSpace, Illumina's customized cloud computing environment that eliminates the need for onsite infrastructure.
  • What is the success rate for NuPCR assays?
    • NuPCR designs are bioinformatically validated in DesignStudio.  Multiple parameters are evaluated to provide the best possible assays.
  • What is the success rate for TruSeq Targeted RNA Expression designs?
    • Illumina internal testing finds that > 90% of non-validated assays correlate with RNA-Seq data. Assays marked validated have been shown to give results consistent with fold changes observed by RNA-Seq in testing across human tissues.
  • What is the support policy for VariantStudio?
    • Our policy for the VariantStudio software is to support the previous software version for 12 months after a new version is released.  The content of the Annotation Database is aggregated from a broad range of external sources, including public and private sources.  At the time of a release for the Annotation Database, the content is locked down and versioned.  By the nature of private annotation sources, our ability to maintain annotation content for the 12-month period may not be possible in rare occurrences.  In these rare occurrences, we make our best efforts to communicate the changes to you as soon as possible to reduce any potential impact on your pipeline processes.

  • What is the temperature resolution of the Eco system?
    • The temperature resolution of the Eco system is 0.1° C.

  • What is the throughput of HiSeq X Ten?
    • Each system can generate 1.8 Tb in less than three days with a daily throughput of about 600 Gb.  This throughput enables 16 genomes covered at 30x per run per system.

  • What is the throughput of the BeadXpress Reader?
    • Throughput is dependent upon the level of multiplexing and whether you are running a single-color or dual-color detection scanner. Typical throughputs are:

      Multiplex

      Single-Color Detection

      Dual-Color Detection

      10

      140 samples/hr

      120 samples/hr

      96

      90 samples/hr

      68 samples/hr

      384

      44 samples/hr

      30 samples/hr

  • What is the typical library size distribution for TruSeq RNA Access libraries?
    • The size of the library distribution of DNA fragments ranges from approximately 200 bp–1 kb, with a final product expected at approximately 260 bp. A larger fragment size is expected for good FFPE RNA (> 350 nt), while a smaller fragment size is expected for poor FFPE RNA. The following is an example:

      TruSeqRNAAccessLibraryDistribution

  • What is the typical library size distribution for my Nextera Rapid Capture Enrichment libraries?
    • Nextera Rapid Capture Enrichment libraries generally range from about 150–1000 bp with the main peak at about 300–350 bp. The following is an example of a post-enrichment (12-plex enrichment) library distribution:

      NexteraRapidCaptureLibraryDistribution

  • What is the typical library size distribution for my final TruSeq Synthetic Long-Read DNA libraries? What is the expected yield?
    • Expected final pooled libraries range from 200–3000 bp when assessed on a Agilent Technologies 2100 Bioanalyzer using a High Sensitivity DNA chip. Illumina has observed a wide range of concentrations (20-200 nM) depending input gDNA quality, operator technique, and other variables.
  • What is the workflow for dual indexing?
    • Please see the appropriate HiSeq instrument user guide for details on the loading of reagents with different workflows and which primers you need to use for your library type.

      • Read 1: An indexed Read 1 follows the standard Read 1 protocol using reagents provided in the TruSeq SBS Kit. The Read 1 sequencing primer is annealed to the template strand during the cluster generation process on the cBot (HP6 or HP10).
      • Index Read 1 (i7): Following the completion of Read 1, the run proceeds to Index Read preparation. The Read 1 product is removed and the Index 1 (i7) sequencing primer (HP8 or HP12) is annealed to the same template strand. The run proceeds through eight cycles of sequencing to read the Index 1 (i7).
      • Index Read 2 (i5): For paired-end flow cells, the Index 1 (i7) Read product is removed and the template anneals to the grafted P5 primer on the surface of the flow cell. The run proceeds through an additional seven chemistry-only cycles (no images are taken) followed by eight cycles of sequencing to read Index 2 (i5).
        For single-read flow cells, the Index 1 (i7) Read product is removed and the Index 2 (i5) sequencing primer (HP9) is annealed to the same template strand. The run proceeds through eight cycles of sequencing to read the Index 2 (i5).
      • Paired End Resynthesis: Read 2 re-synthesis for dual-indexed paired-end sequencing uses reagents provided in the TruSeq PE Cluster Kit.
      • Read 2: Read 2 follows the standard paired-end sequencing protocol using standard SBS reagents.
  • What is this product?
    • TruSeq Custom Amplicon (TSCA) is the fastest and easiest multiplexed amplicon assay optimized for the MiSeq System. It allows for the sequencing of 16–1536 custom amplicons (small regions of interest) across up to 96 samples. The assay also includes on-instrument variant calling for both germline and somatic variation with the MiSeq System.
  • What kind of data can I export from KaryoStudio?
    • You can export data displayed in the Found Regions table as either a single row or the entire table.

      • To export a single row: right click in the Found Regions table and select Copy Row to Clipboard.
      • To export the whole table: right-click in the Found Regions table and select Copy All to Clipboard
      • Paste the data into an Excel file or import it into another downstream, third-party application. In addition, you can export data in the form of a cytogenetics report.

      There are no other reporting functions available in KaryoStudio.

  • What kind of data quality can I expect from a run on the HiSeq X?
    • You can expect greater than 75% of bases above Q30 with a 2 x 150 bp run using a human library prepared with the TruSeq DNA Nano HT Sample Prep Kit (350 bp insert) or an Illumina PhiX control library.

  • What kind of flow cells and reagents are supported on HiSeq X Ten?
    • HiSeq X is the first platform using patterned flow cell technology. Reagents provided in teh HiSeq X HD Reagent Kit are specifically designed and optimized for HiSeq X Ten. Non-patterned flow cells and SBS kits designed for other HiSeq models have not been validated and are not supported for use on the HiSeq X.

  • What kind of quality scoring method does Illumina use?
    • A quality score (or Q-score) is a  prediction of the probability of an incorrect base call. Based on the Phred scale, the Q-score serves as a compact way to communicate very small error probabilities. Given a base call, X, the probability that X is not true, P(~X), is expressed by a quality score, Q(X), according to the relationship:
      Q(X) = -10 log10(P(~X))
      where P(~X) is the estimated probability of the base call being wrong.

      A quality score of 10 indicates an error probability of 0.1, a quality score of 20  indicates an error probability of 0.01, a quality score of 30 indicates an error probability of 0.001, and so on.

      During analysis, base call quality scores are written to FASTQ files in an encoded compact form, which uses only one byte per quality value. This method represents the quality score with an ASCII code equal to the value + 33.

  • What kind of support is provided for ChIP-Seq?
    • We provide the same support as for standard genomic DNA resequencing. However, we do not provide support on antibodies or the ChIP portion.


  • What kit sizes are available for sequencing on the NextSeq 500?
    • NextSeq 500 kits are available in three sizes: 300 cycles (2 x 150), 150 cycles (2 x 75), and 75 cycles (1 x 75). For output specifications, see NextSeq 500 specifications page

  • What level of indexing is supported for TruSeq DNA and RNA Sample Prep v1 and v2/LT?
      • TruSeq DNA and RNA Sample Prep v1 kits support 12-plex per lane and 96-plex per flow cell.
      • TruSeq DNA v2/LT and RNA Sample Prep v2 kits support 24-plex per lane and 192-plex per flow cell.
  • What level of indexing is supported for TruSeq Small RNA?
    • TruSeq Small RNA Sample Preparation Kits currently support 48-plex per lane.

  • What level of indexing or barcoding is available?
    • Up to 96-plex indexing is supported for TruSeq Custom Amplicon, Nextera, Nextera XT, and TruSeq DNA Sample Prep Kits.

  • What level of multiplexing is supported on the GA with Nextera? When will this be available?
    • There will be 96 available barcodes using 12x8 indices, combining 12 Index 1(i7) with 8 Index 2 (i5) indices. Although kits are currently available, (the index kit supports multiplexing on all Illumina sequencing platforms) multiplexing on GA is not supported until updated SCS/RTA software is available.
  • What level of multiplexing is supported on the HiSeq with Nextera?
    • There are 96 available barcodes using 12x8 indices, combining 12 Index 1(i7) with 8 Index 2 (i5) indices.
  • What level of multiplexing is supported on the MiSeq with Nextera?
    • There are 96 available barcodes using 12x8 indices, combining 12 Index 1(i7) with 8 Index 2 (i5) indices.

  • What level of sample plexity is supported for TruSeq Exome Enrichment?
  • What mathematical model does the Illumina Eco analysis software use for relative quantification experiments?
    • The Illumina Eco analysis software uses the ΔΔCq method (Livak et al., 2001), which is the most commonly used analysis method for relative quantification. The ΔΔCq method reports fold change in gene expression of a target gene relative to that of a reference gene and reference (calibrator) sample. The reference gene is typically a housekeeping gene such as GAPDH, β-actin, β2-microglobin or HPRT. The choice of reference sample can vary depending on the type of gene expression experiment. For example, a reference sample might be an untreated control sample relative to a treated sample, normal tissue relative to diseased tissue, or liver relative to brain.

      For the ΔΔCq method to be valid certain assumptions must be met. First, the reference gene must be stably expressed between different cells of tissues and unaffected by any experimental treatment. Second, the amplification efficiencies of both target gene and reference gene must be approximately equal. Both of the conditions should be experimentally validated for each experiment before the ΔΔCq method can be used.

      When extreme accuracy is needed, the Eco software supports use of multiple reference genes as well as PCR efficiency correction.

  • What method do I use to quantify my input genomic DNA for the TruSight One
    • Illumina recommends using a fluorometric-based method that is specific to double-stranded DNA, such as QuantiFluor or PicoGreen. Consult the DNA Input Recommendations section of the TruSight One Library Prep Guide for additional information.

  • What method should I use to quantify my Nextera Rapid Capture Enrichment tagmented library before sample pooling?
    • Illumina recommends quantifying the tagmented library using a fluorometric-based method specific for double-stranded DNA, such as QuantiFluor or PicoGreen. For more information, see the DNA Input Recommendations section of the Nextera Rapid Capture Enrichment Guide .
  • What method should I use to quantify my enriched Nextera Rapid Capture Enrichment library?
    • Illumina recommends quantifying the enriched library using a fluorometric-based method specific to double-stranded DNA, such as QuantiFluor or PicoGreen. For more information, see the Validate Library section of the Nextera Rapid Capture Enrichment Guide.

      qPCR can also be used to quantify the final enriched Nextera Rapid Capture Enrichment library. For more information, see the Sequencing Library qPCR Quantification Guide.

  • What method should I use to quantify my final TruSeq Synthetic Long-Read DNA libraries?
    • Quantify your libraries using a fluorometric quantification method that uses dsDNA binding dyes or qPCR.
  • What method should I use to quantify my input genomic DNA for TruSeq Synthetic Long-Read DNA library prep?
    • Illumina recommends using fluorometric based methods for quantification, such as Qubit or PicoGreen, to provide accurate quantification for dsDNA. UV spectrophotometric based methods, such as the Nanodrop, measure any nucleotides present in the sample including RNA, dsDNA, ssDNA, and free nucleotides. This can give an inaccurate measurement of gDNA.
  • What method should I use to quantify my input genomic DNA for the Nextera Rapid Capture Enrichment protocol?
    • Illumina recommends using a fluorometric-based method that is specific to double-stranded DNA, such as QuantiFluor or PicoGreen. For more information, see the DNA Input Recommendations section of the Nextera Rapid Capture Enrichment Guide .
  • What method should I use to quantify my total RNA input for the TruSeq RNA Access Library Prep protocol?
    • Illumina recommends using RiboGreen or Qubit to accurately quantify the total RNA input. For more information, see the RNA Input Recommendations in the TruSeq RNA Access Library Prep Guide .
  • What methods does does Illumina recommend for quantifying ChIP DNA starting material for TruSeq ChIP sample prep?
    • Illumina recommends using fluorometric based methods for quantification, including Qubit or PicoGreen to provide accurate quantification of ChIP DNA. UV-spec based methods, such as the Nanodrop, will measure any nucleotides present in the sample including RNA, dsDNA, ssDNA, and free nucleotides which can give an inaccurate measurement of ChIP DNA.

  • What methods of shearing are supported by TruSeq DNA PCR-Free Sample Prep?
    • The protocol is optimized for shearing using Covaris fragmentation. Other methods are not supported or tested by Illumina and may result in low yield, unexpected size distributions, or sample failure.
  • What methods of shearing are supported by TruSeq DNA Sample Prep?
    • Illumina recommends Covaris shearing, but testing with nebulization shows comparable data.

  • What methods of shearing are supported by TruSeq Nano DNA Sample Prep?
    • The protocol is optimized for shearing using Covaris fragmentation. Other methods are not supported or tested by Illumina and may result in low yield, unexpected size distributions, or sample failure.
  • What molecules are removed with the TruSeq Stranded Total RNA Sample Prep Kit with Ribo-Zero Globin?
    • The Ribo-Zero removal mix targets cytoplasmic rRNA, mitochondrial rRNA, plus globin mRNA.
  • What molecules are removed with the TruSeq Stranded Total RNA Sample Prep Kit with Ribo-Zero Plant?
    • The Ribo-Zero removal mix targets cytoplasmic (25S, 18S, 5.8S, and 5S rRNA), Chloroplast (23 S, 16S, 5S, and 4.5S), and mitochondrial rRNA (18S and 5S).
  • What molecules are sequenced with the TruSeq Stranded mRNA Sample Prep Kit?
    • All poly-adenylated molecules are isolated and sequenced. There are species of non-coding RNA that are poly-adenylated as well.
  • What operating system does the NextSeq 500 use?
    • The operating system on the NextSeq computer is Windows 7.
  • What operating system is used on the MiSeq?
    • The MiSeq runs on Windows 7 Embedded Standard.

  • What operating system is used on the instrument computer?
    • The HiSeq instrument computer employs 64-bit Windows Vista.
  • What organisms do the TruSeq Stranded Total RNA Sample Prep kits support?
    • The TruSeq Stranded Total RNA Sample Prep kits with Ribo-Zero Human/Mouse/Rat and TruSeq Stranded Total RNA Sample Prep kits with Ribo-Zero Gold both support human, mouse, and rat organisms. That use of these kits with other organisms has not been tested and is not supported.

  • What peripheral equipment is needed to perform the Illumina Nextera sample prep?
    • A standard thermocycler is required to perform PCR.
  • What ports, domains and encryption does BaseSpace use?
    • Contact your local IT administrator if local security policies have to be modified to allow access to BaseSpace. BaseSpace uses SSL/https port 443 and the domains *.basespace.illumina.com and *.s3.amazonaws.com. Data streaming to BaseSpace is encrypted using the AES256 standard and uses SSL for protection. More information on encryption can be found at http://blog.basespace.illumina.com/2011/12/13/basespace-security/
  • What power connections are required?
    • The NextSeq instrument comes with a region-specific power cord. For more information, see the NextSeq 500 System Site Preparation Guide .
  • What probe pool is used in TruSeq RNA Access Library Prep?
    • Coding Exome Oligos (CEX) are used, which are also used in Nextera Rapid Capture Exome Enrichment.
  • What programs can I install on the HiSeq instrument computer?
    • The instrument computer is a computational engine performing real time analysis of data. To avoid loss of data and other adverse effects, Illumina does not recommend installing any additional software with the exception of anti-virus software.

  • What quality control measures are in place before, during, and after the genotyping services?
    • Throughout our process, we have developed quality control procedures to ensure you with the highest quality of data possible. These include (1) quality control and quantification of incoming DNAs, (2) multiple internal controls built into each genotyping assay, (3) barcoded labeling of sample plates, (4) error-free sample tracking under active database control to provide error-free handling of samples, assays and data (PosiTrack), and (5) statistical measures of success for assay development and genotyping confidence scores (GenCall).
  • What quality of DNA is required and how should this be assessed?
    • Commercially available or laboratory validated DNA extraction methods typically yield DNA that is compatible with this assay. DNA purity should have an A260/A280 ratio of 1.8–2.0. PicoGreen is recommended. If using FFPE DNA at the supported amplicon sizes (150 or 175bp amplicons), you will need to follow the recommended QC steps to select DNA of appropriate quality.
  • What quality of DNA is required for the TruSight DNA Amplicon assay and how should this be assessed?
    • Commercially available or laboratory validated DNA extraction methods typically yield DNA that is compatible with this assay. DNA purity should have an A260/A280 ratio of 1.8—2.0. PicoGreen is recommended.
  • What quantitation methods does Illumina recommend for final library quantitation?
    • Double Stranded DNA specific dyes, such as QuBit or picogreen.
  • What quantitation methods does Illumina recommend for final library quantitation?
    • Illumina recommends quantifying your TruSeq ChIP libraries using qPCR according to the Illumina Sequencing Library qPCR Quantification Guide.

  • What quantitation methods does Illumina recommend for starting material?
    • Double Stranded DNA specific dyes, such as QuBit or picogreen.
  • What reagents do I need to perform bisulfite sequencing applications?
  • What sample prep do I use for MiSeq?
  • What sample preparation kits are compatible with TruSeq Enrichment Kits?
    • For exome or custom enrichment sequencing, TruSeq DNA Sample Prep Kits must be used.

  • What sequencing primers are needed for TruSeq RNA Access libraries?
    • Standard sequencing primers included in the cluster generation kits are required.
  • What sequencing primers are needed for TruSeq v2/LT or HT kit sample prep?
    • For TruSeq v2/LT kits, standard sequencing primers included in the cluster generation kits are required.

      For TruSeq HT kits, primer usage will depend on the flow cell type used. Primers in the TruSeq Dual Index Sequencing primer kit, single read (catalog # FC-121-1003) are needed to run TruSeq HT Dual-Indexed libraries on single read v3 flow cells.  These primers are HP10 (read 1), HP12 (Index read 1), HP9 (Index read 2 for single read flow cells only).  This add-on box is not required if sequencing a TruSeq HT prepared library with the MiSeq System or on single-read rapid flow cells, or paired-end v3 or Rapid flow cells for HiSeq, HiScanSQ, GAIIx. The primers in the TruSeq Dual Index Sequencing Primer kits are backwards compatible with all Illumina libraries; there is no need to spike in primers as these primers are backwards compatible with other Illumina library types.

  • What sequencing primers do I need to sequence my TruSeq Synthetic Long-Read DNA libraries?
    • Use the TruSeq Dual Index Sequencing Primer Box, Paired-End (Catalog # PE- 121-1003) when performing cluster generation with the TruSeq PE Cluster Kit v3. If you are using TruSeq PE Rapid Cluster kit or HiSeq PE Cluster Kit v4, no additional primers are required.
  • What sequencing primers should I use for TruSeq ChIP libraries?
    • TruSeq ChIP libraries are compatible with sequencing primers included in all TruSeq cluster kits as well as in TruSeq Dual Index Sequencing Primer Box, Single Read or Paired End.

  • What sequencing read length is supported on the HiSeq X system?
    • Supported read lengths for the HiSeq X are up to 2 x 150 bp plus single 8 bp indexing.

  • What sequencing, and indexing reagent combinations are compatible with the TruSeq Nano DNA Sample Prep kits?
    • Dual index sequencing of TruSeq Nano DNA HT libraries on the HiSeq, HiScanSQ, or GAIIx system using single-read flow cells requires the TruSeq Dual Index Sequencing Primer Box, Single Read (FC-121-1003) to be ordered separately. This add-on box is not required if sequencing a TruSeq Nano DNA HT prepared library with the MiSeq System, HiSeq Rapid two-lane single or paired-end flow cells, or on traditional paired-end flow cells for HiSeq, HiScanSQ, GAIIx. Additional SBS reagents may be necessary to account for all index cycles.
  • What should I do if I get an error indicating that the program can't be found? For example: -bash-3.1$ GAPipeline-1.3.2/bin/illumina2srf -o lane_1.srf / /Data/IPAR_1.3/Bustard1.3.2_01-03-2009/s_1_*_qseq.txt -bash: /GAPipeline-1.3.2/bin/illumina2srf: No such file or directory
    • If you get this message, you should explicitly install the io_lib. To do this, run the following command from the Pipeline Install directory: /GAPipeline_1.3.2 make WITH_IO_LIB=1 install, then retry the command from Q2, above.
  • What should I do if the copy speed is too slow?
    • If you are not copying files to the target server fast enough, consider the following:

      • Illumina recommends a network connection rated at 1 Gigabit or faster. Ask your IT department to upgrade your network.
      • If the target server is nearly full, copying takes much longer. Remove files that are no longer needed from the target server, or copy to a different server.
      • If multiple sequencing instruments copy to the same server, the server drive might not be fast enough to handle the traffic. Consider setting up the instruments to copy to different servers.
      • If multiple sequencing instruments share a connection to the target server, the connection might not be fast enough to handle the traffic. Consider setting up the instruments to use different routes.
  • What should I do if there is an issue with the Seq part of my ChIP-Seq?
    • Please refer to the standard protocols for the resequencing of genomic DNA.
  • What size DNA does Illumina recommend for the Infinium Methylation Assay?
    • Similar to the Infinium genotyping assay, we recommend DNA that is sized ≥ 2kb, which can be assessed on an agarose gel.

  • What software do I use to analyze cifs generated with HCS 2.x?
    • You can use OLB 1.9.4. However, please note that OLB 1.9.4 will be replaced by RTAOLB, which is a means of running RTA in a Linux environment. An RPM of RTA plus needed libraries and instructions will be released on My Illumina in November of 2012.
  • What software does Illumina offer for analysis of TruSeq RNA Access libraries?
    • Illumina offers the BaseSpace Core Apps for RNA. Learn more about BaseSpace .
  • What software is needed to run TruSeq Synthetic Long Read libraries?
      • IEM 1.8 or later is required to create a sample sheet.
      • cBot 1.4 software or later (v3) or cBot 2.0.1 or later (v4) may be needed for cluster generation.
      • HCS 2.0/RTA 1.17 or later is required for sequencing.
      • The BaseSpace TruSeq Long-Read Assembly App or TruSeq Phasing Analysis App are required for analysis.
  • What software is required for sequencing dual-indexed libraries?
    • HiSeq users must upgrade to HCS1.5/RTA1.13 or higher and GA users must upgrade to SCS2.10/RTA1.13 or higher.

      Category

      Software Version

      Instruments

       Required

      Instrument Software and Sequencing Run QC

      HCS 2.0/RTA 1.17.20/SAV 1.8.20/Recipe Fragment 1.3.54

       

      HiSeq/HiScanSQ

      Yes

      SCS 2.10/RTA 1.13/SAV 1.8.20

      GA

      Yes

      Analysis

      CASAVA 1.8.2

       

      HiSeq/HiScanSQ/GA

      Yes

      Sample Sheet Creation

      Illumina Experiement Manager 1.3

      HiSeq/HiScanSQ/GA

      Recommended

  • What software is required/available to support Nextera dual-indexing? Which versions?
    • Software Component/version:

      • HCS 1.5/RTA 1.13/SAV 1.8.4
      • SCS 2.10/RTA 1.13/SAV 1.8.4
      • MiSeq Reporter 1.1
      • CASAVA 1.8.2
      • Illumina Experiment Manager 1.0

      Please note that SCS 2.10/RTA 1.13 for GA is scheduled to be available November 2011.

  • What software should be used for analysis of TruSeq stranded mRNA libraries?
    • Demultiplexing of dual-indexed sequencing runs requires CASAVA1.8.2. Alignment of single read runs may be performed using Casava 1.8.2. For alignment of paired-end runs, Illumina recommends TopHat/Cufflinks. For more information,  see the RNA Sequencing Analysis With TopHat guide. TopHat is not an Illumina supported product, but an open source initiative. Additional, third party analysis tools are also available. Learn more about Illumina's RNA applications .
  • What software versions are required to sequence my Nextera Rapid Capture Enrichment prepared libraries?
      • IEM: Illumina Experiment Manager 1.5 software or later
      • cBot software (if needed): cBot 1.4 software or later
      • HCS software for HiSeq/HiScanSQ: HCS 1.5/RTA 1.13 software or later
      • SCS software for Genome Analyzer: Genome Analyzer SCS 2.10/RTA 1.13 software or later
      • HiSeq Analysis Software (HAS): any version
      • MiSeq Control Software (MCS): 2.2 software or later
      • MiSeq Reporter Software (MSR): 2.2 software or later
  • What standard panels are available for GoldenGate genotyping?
    • LinkageIVb, Mouse LD Linkage, Mouse MD Linkage, MHC Panel Set, Cancer Panel, DNA Test Panel
  • What steps are automated in the GoldenGate Assay for Methylation protocol?
    • The automated processes specific to GoldenGate Assay for Methylation are:

      • Make BCS/BCM
      • Precipitate BCS/BCM
      • Resuspend BCS/BCN
      • Make ASE

      The remaining steps are identical to genotyping using the GoldenGate Assay.

  • What strand is sequenced in the TruSeq Stranded RNA sample prep kits?
    • The cDNA strand is sequenced.

  • What technologies does Illumina use in its Genotyping Services business?
    • We have created a highly multiplexed SNP genotyping system using our proprietary BeadArray technology. In addition, we have developed a fail-safe sample-tracking LIMS system to ensure error-free processing.
  • What third party software can be used for analysis of TruSeq RNA Access libraries?
    • Illumina's BaseSpace Core Apps for RNA includes the industry standard TopHat and Cufflinks analysis pipeline. Additional, third party analysis tools are also available.
  • What tools are offered for data analysis with TruSeq Custom Amplicon?
    • The following software tools are available for TruSeq Custom Amplicon: Illumina Experiment Manager, MiSeq Reporter, and the Illumina Amplicon Viewer. The MiSeq Reporter is simple, on-instrument software that demultiplexes indices/samples, performs alignment of reads, and creates a variant report immediately upon completion of each sequencing run. The MiSeq Reporter also creates an assembly for each amplicon (on a per sample basis), per SNP calling, and generates graphs and reports including the sequencing coverage per amplicon. The Illumina Amplicon Viewer performs data visualization from multiple runs and custom report generation off of the instrument. Analysis workflows are available for detecting both germline and somatic variation in sequenced samples.
  • What tools are offered for data analysis with TruSight DNA Amplicon?
    • The following software tools are available for TruSight DNA Amplicon: Illumina Experiment Manager, MiSeq Reporter, Illumina Amplicon Viewer, and VariantStudio. The MiSeq Reporter is simple, on-instrument software that demultiplexes indexes and samples, performs alignment of reads, and creates a variant report immediately upon completion of each sequencing run. The MiSeq Reporter also creates an assembly for each amplicon (on a per sample basis), performs SNP calling, and generates graphs and reports including the sequencing coverage per amplicon. The Illumina Amplicon Viewer performs data visualization from multiple runs and custom report generation off the instrument.

      The Illumina VariantStudio desktop application is available with purchase of TruSight panels and includes a one-year license to the VariantStudio desktop application. VariantStudio imports variant call files generated during analysis of sequencing data and provides commands to annotate variants. VariantStudio filters results using various filtering options, classifies variants according to their biological impact, and exports results to a report.
  • What total-RNA quantification method do you recommend?
    • Any routine method is fine. Illumina recommends using RiboGreen to quantify total-RNA concentration.
  • What type of data files are output from TruSeq RNA Access library data analysis?
  • What type of information should I provide when contacting Technical Support?
    • For the fastest and most efficient support, supply your name, institution, contact information, and the serial number from the label on the back of your instrument. The warranty, instrument, and previous case history are associated with the instrument serial number, so this information enables Technical Support scientists to obtain the data they need to support you.

      If you are contacting Technical Support concerning run data, please also provide the InterOp folder, runparameter.xml file, and runinfo.xml file.

  • What type of sample prep is supported on the HiSeq X system?
    • For runs on the HiSeq X, Illumina supports the TruSeq DNA Nano HT sample prep kit (catalog # FC-121-4003) with 350 bp inserts.
  • What types of assays can be developed with the VeraCode Beads?
    • The glass surface of the VeraCode beads make them ideal for a number of bioassays, including genotyping, gene expression, methylation, and protein-based assays. Solution-based assays, in conjunction with microarrays, can also be developed.
  • What types of controls should be run for a ChIP assay?
    • Common controls include no primary antibody or use of normal rabbit IgG in ChIP as a negative control. In most cases, if you are studying the same factor in different tissues or cell lines, one control is sufficient. More controls will be needed if you are studying multiple factors from the same source.

      We have seen researchers use either a genomic DNA input control or a "mock" control with IgG. A mock sample is usually generated by the following process: after sonication, divide your sample into two equal parts and add your antibody to one part, while adding mock antibody (normal IgG) or pre-immune serum to the other. The goal is to ensure that you are not seeing sequences that stick non-specifically to the antibodies and that no other part of the process is influencing your results (a process control). In addition, it ensures that the recognition of your protein by the antibody you are using is required for enrichment of the target sequence. We have received feedback from several successful ChIP-Seq researchers who have told us that a mock control is the best, with the lowest background, but this does require more work than an input control.

  • What types of inputs are supported by ADT?
    • Accepted inputs include RSList, GeneList, SequenceList, and RegionList.
  • What types of target sequence inputs are accepted in DesignStudio when creating a TruSeq Custom Amplicon project?
    • The following inputs are accepted in DesignStudio: chromosome number with location of starts and stops, gene name (with an option for exons only as well as up- and down-stream bases), and batch upload with a .csv file. Templates for coordinates and genes are provided in the file upload section for adding new target regions. For additional information, refer to the online help functionality in DesignStudio.
  • What version USB is on the MiSeq?
    • The MiSeq is equipped with USB 2.0.
  • What warranty is provided with the Eco system?
    • The Eco system comes with a one-year warranty that provides full coverage for all service parts and labor, hardware and software updates, and access to online training modules. The warranty includes comprehensive email and telephone support for the instrument, its applications, and its bioinformatics. Service is conducted at the manufacturer site and will be completed within five business days on average.

      Extended warranty and calibration plans are available if needed.

  • What will I need (reagents, etc.) to run the Infinium OncoArray-500K?
    • Customers will receive all reagents necessary to run the BeadChips included in their order. For robust results, Illumina recommends a minimum of 200 ng of DNA at a minimum concentration of 50 ng/μl (as measured by a fluorescent method of quantification). Access to an Illumina iScan or HiScan system is required for processing the BeadChips. Information on service providers and core facilities can be found at http://www.illumina.com/services.ilmn, or by contacting your local account manager.

  • What will happen if I use less than 1 µg of total RNA?
    • The overall yield and quality of the library can diminish with lower input amounts. The PCR cycles in the enrichment step of the protocol (following the gel) are tuned for a certain amount of input material. The protocol calls for exactly 15 cycles of PCR amplification. If the amount of material purified from the gel is significantly less than the standard 1–10 µg prep, then you might need more PCR cycles to create a clearly visible band. However, we do not recommend over amplifying the library, since this can lead to artifacts during the electrophoresis and quantification of the library.
  • What workflow do I use in the Illumina Experiment Manager for creating a sample sheet for my Nextera Rapid Capture Enrichment project?
    • Use the Enrichment workflow to create a sample sheet formatted for analysis of Nextera Rapid Capture Enrichment data. For more information, see the IEM Nextera or TruSight Quick Reference Card for instructions on creating your sample sheet.
  • What’s new in the GenomeStudio microarray modules (GT, GX, M, and PT v1.0)?
    • Please see the GenomeStudio Software 2009.2 Release Notes, available in iCom and the GenomeStudio Portal, for details.

  • When I downloaded the iControlDB program, I got the Final Case Report template files but the product I am interested in is not contained in the files. How can I get this file?
    • Please contact TechSupport@illumina.com and they will forward you files for any recent products.
  • When I right click the mouse on the samples table, I see an option that says “Request to be Admin”. When I select this, it says I do not have permission. What is this function?
    • Admin rights are only available for Illumina personnel who are adminstrators for the iControlDB.
  • When I started my run, I got the HCS error message, “Must have at least one valid ETF to normalize/correct the failed ones.” What does this mean and what should I do?
    • This error message indicates a lack of fluorescence on the flow cell. To find focus at the start of a run, the software uses ETF, which is a focusing method that reads fluorescence from clusters on the flow cell. ETF must find fluorescence in at least one lane of the flow cell before the run can begin.

      To correct this problem, perform a primer rehybridization. Re-annealing the Read 1 sequencing primer usually increases the fluorescence if clusters are present on the flow cell. Additionally, check the cBot plate to make sure that all reagents were delivered correctly and that the sequencing primer was appropriate for your library types. When you reload the flow cell on the HiSeq, confirm that the fluidics system is functioning correctly. If a primer rehybridization does not resolve the issue, contact Illumina Technical Support for further assistance.

  • When changing parameters in the Clustering Options dialog box, will this impact or change the existing clustered data?
    • No, the new settings will only be applied to any SNPs clustered after applying changes.
  • When did the new MCS4 plus RTE formulation ship?
    • The new MCS4 plus RTE formulation started shipping in late April, 2012. All new orders were automatically shipped with the new material. There is little or no inventory left of MCS3.
  • When dual-indexing, if I use mismatches=1 for CASAVA, will it take into account 1 mismatch per index or only 1 mismatch total across both indices?
    • It will take into account one mismatch per index. The argument in CASAVA is a comma-delimited list of number of mismatches allowed for each read (for example: 1,1). If a single value is provided, all index reads will allow the same number mismatches. the default is 0. The index reads are treated completely separately.

  • When generating a sample report, can I import sample information instead of entering it manually into VariantStudio?
    • Yes. When you generate a report from the Sample Report menu, you can either type in the sample information requested in the chosen report template or import the sample information from an external text file.

  • When generating fewer than the full amount of samples supported by a TruSeq HT sample prep kit, how should the core reagents be aliquoted or freeze/thawed?
    • The HT kit provides sufficient reagents for 96 total sample preparations, in two tubes of 48 sample preparations each. If you do not intend to consume the entire reagent in a tube in one use, we recommend dispensing the reagent into aliquots (a minimum of 24 sample preps/aliquot) after the initial thaw, and refreeze the remaining aliquots to avoid excessive freeze/thaw cycles. However, if you aliquot, you may not have enough reagents for the full number of reactions over multiple uses. When preparing less than 24 samples at a time, Illumina strongly recommends using the LT kit.
  • When is analysis of a sequencing run performed?
    • Image analysis occurs in real time, phasing estimates and base calling begin occur after cycle 12, and base call quality scoring occurs after cycle 25.
  • When is it best to use Nextera Rapid Capture Custom Enrichment vs. TruSeq Custom Amplicon for my custom target projects?
    • A primary determinant is the total size of the target regions of interest. In general, smaller custom designs (up to ~0.5 Mb) are optimal for TruSeq Custom Amplicon based approaches and are best suited for sequencing on the MiSeq system. Nextera Rapid Capture Custom Enrichment is a better match for mid-to-large custom designs (~0.5–15 Mb) and may pair best with a HiSeq or MiSeq system.
  • When should I choose Nextera Enrichment vs. TruSeq Enrichment?
    • Nextera Enrichment is optimized for a low DNA input requirement and utilizes the Nextera technology for a faster workflow. TruSeq Enrichment offers the highest levels of coverage uniformity.
  • When should I use a dedicated PhiX control lane for sequencing?
    • For samples with an unbalanced base composition, Illumina recommends using a dedicated PhiX control lane to improve cross-talk and phasing calculations. Unbalanced samples contain genomes with AT or GC content of less than 40% or greater than 60%. PhiX Control v3, Catalog # FC-110-3001, is a balanced and diverse library with a mean insert size of approximately 375 bp, corresponding to 500 bp library size if visualized on a Bioanalyzer.

      Low diversity libraries, where a significant number of the reads have the same sequence, require a dedicated control lane and a high-concentration spike-in (40% or higher) to create a more diverse set of clusters. For more information, see Using a PhiX Control for HiSeq Sequencing Runs.

  • When using HiSeq v4 reagents, do you have to change reagents during the run as with TruSeq v3 reagents?
    • HiSeq v4 kits are designed for a walk-away workflow, which means that all reagents are loaded before starting the run. Splitting incorporation buffer for Read 2 is no longer necessary.
  • When will I be invoiced?
    • Customers will be invoiced upon shipment.
  • When will quality scores appear during a sequencing run on the MiSeq?
    • Quality scores appear after cycle 25. The first 25 cycles are used to determine the chastity of a base call, which in turn determines the quality filter.
  • Where are data stored after they are imported into VariantStudio?
    • The desktop version of Illumina VariantStudio stores data locally in the VariantStudio project file. During the import of VCF files, data are loaded to the desktop memory. To save the data, designate a location to save the project, which can be reopened later.

  • Where are the content manifest file downloads for my Nextera Rapid Capture Custom Enrichment assay pool?
    • Target and Probe manifest files can be downloaded from the following locations online:
      • The Export Manifest function in Project Dashboard of your DesignStudio project
      • The Product Files section of in Custom Products in your MyIllumina account
  • Where are the content manifest file downloads for my TruSeq Targetetd RNA Expression assay pool?
    • Manifest files can be downloaded using the Export Manifest function in Project Dashboard of your DesignStudio project. The manifest file becomes available once your order ships, not when the order is placed or designed in DesignStudio.
  • Where can I find Safety Data Sheet (formally MSDS) information for the TruSight One kits?
    • Email Illumina Technical Support to receive the TruSight One SDS (MSDS) documents.

  • Where can I find a marker list of all the CpG sites on the Infinium HumanMethylation450 BeadChip?
    • The manifest for the Infinium HumanMethylation450 BeadChip can be found in our online store .
  • Where can I find information about integrating probes on the HumanMethylation450 BeadChip with those on the HumanHT-12 Expression BeadChip?
  • Where can I find my GenomeStudio software license keys?
    • The 16-digit license keys may be found in the confirmation of your sales order associated with the purchase of GenomeStudio and the purchase of your instrument. Each GenomeStudio module has an individual key.
  • Where can I find technical information describing HiSeq v4 data compression options?
  • Where can I find the manifest for TruSight One?
    • The TruSight One manifest can be downloaded from the TruSight One Product Support page on illumina.com.

  • Where can I find the probe coordinates for the probes in the Human and Mouse MAPs?
    • Illumina has the probe coordinates (i.e., chromosomal location information) for most of the miRNA, including those not in the Sanger miRBase. You can find this information in the *.bgx file. See http://www.switchtoi.com/annotationfiles.ilmn for more information.
  • Where can I find the source code for the Isaac algorithms? Are they “open source”?
    • https://github.com/sequencing. The software is released is under Illumina Open Source Software License available on https://github.com/sequencing/licenses/.The terms of the license are intended to make the software accessible to a wide variety of bioinformatics developers, computational biologists, and other users in the research community, and to encourage community development of the software. For questions about the license or its applicability, please contact Isaac-admin@illumina.com.

      Where can I find more technical details on Isaac and the HiSeq Analysis Software?

      The HiSeq Analysis User Guide contains instructions on the installation and usage of the HiSeq Analysis Software package. You can download the User Guide at http://supportres.illumina.com/documents/documentation/software_documentation/has/has_userguide_15041353b.pdf

      A white paper providing additional technical information about the Isaac aligner and variant caller  is available on the Illumina website: http://res.illumina.com/documents/products/whitepapers/whitepaper_iassc_workflow.pdf

      A peer-reviewed application note is available on the Bioinformatics website:

      Raczy C, Petrovski R, Saunders CT, Chorny I, Kruglyak S, et al. (2013) Isaac: Ultra-fast whole genome secondary analysis on Illumina sequencing platforms. Bioinformatics 10.1093/bioinformatics/btt314 

  • Where can I find workflows for the GenomeStudio sequencing modules?
    • You can find workflow documents for the GenomeStudio ChIP Sequencing, DNA Sequencing, and RNA Sequencing modules in the GenomeStudio Portal or on the Product Documentation web page.
  • Where can I get the OMA manifest file for the GoldenGate Methylation Cancer Panel I?
    • Log onto iCom, our eCommerce site on Illumina's website, and click Downloads. All of the product support files, including the OMA manifest, can be downloaded from this location.
  • Where can I get the adapter, primer, and index sequences used in the TruSeq sample prep kits?
    • The adapter sequences are included in the Illumina Customer Sequence Letter. Additionally, the index sequences are available in the user guide for the TruSeq sample prep kit that you are using. Please note that the oligos are not sold separately. Primer sequences are proprietary.

  • Where do I find the Nextera Rapid Capture Enrichment index sequences to enter into my sample sheet?
    • The Dual Indexing Principle section of the Nextera Rapid Capture Enrichment Guide contains a list of index sequences uses in the Nextera Rapid Capture Enrichment kits.

      Alternately, Illumina Experiment Manager (IEM) software can be used to create your sample sheet and assign correct index sequences. The IEM software can be downloaded from the Nextera Rapid Capture Exome and Expanded Exome Enrichment Kits Downloads or Nextera Rapid Capture Custom Enrichment Kits Downloads support pages.

  • Where do I load my samples on the cBot if I am using the TruSeq Rapid Duo cBot Sample loading kit?
    • Load 135 μl of the denatured and HT1 diluted library into wells 1 and 2 of an eight-tube strip. Then load the eight-tube strip into the cBot tube strip holder, making sure to position wells 1 and 2 towards the right side.
  • Where do I load the 8-port and 10-port gaskets with a two-lane Rapid flow cell and eight-lane high output flow cell?
    • For setting up either a TruSeq Rapid mode, TruSeq v3 mode, or HiSeq v4 mode on a HiSeq 2500 or HiSeq 1500, load the 8-port gasket in the back manifold position and load the 10-port gasket in the front manifold position. For a run on a HiSeq 2000, load 8-port gaskets in both the front and back positions.
  • Where is the control lane feature?
    • The option to designate a control lane has been removed in HCS v2.2. The software includes optimizations that improve the handling of low-diversity libraries, which eliminates the need for a control lane for matrix and phasing estimates.
  • Where is the information in the Classification Database stored?
    • By default, the Classification Database is saved locally in C:\ProgramData\Illumina\Illumina VariantStudio\ClasificationDb.bin. When the database is stored locally, classifications are available for the current project and any future projects opened on that computer. The Classification Database can also be configured to store on a user-specified network location. If the classification database is stored on a network location, classifications are available to projects opened in any installation of VariantStudio with access to that network location.

  • Where is the status.htm file?
    • This report is not created by the MiSeq. However, similar information can be viewed using Sequencing Analysis Viewer (SAV).
  • Which Illumina sequencers are compatible with TruSight One libraries?
    • TruSight One libraries can be run on all Illumina sequencing systems. A trio of TruSight One-enriched sample libraries can be sequenced so that >95% of the target regions are covered at 20x minimum coverage. This coverage can be achieved using the supplied 300-cycle MiSeq v3 reagent cartridge included as part of the 9-sample TruSight One kit. The 36-sample TruSight One kit does not contain sequencing reagents. Lack of reagents allows flexibility to prepare 12-plex pooled sample libraries that can be sequenced to target depths with a single flow cell using the HiSeq2500 in rapid run mode.
  • Which Illumina sequencing instruments are compatible Nextera Rapid Capture Enrichment sample prep?
    • Nextera Rapid Capture Enrichment libraries can be run on all Illumina sequencing systems; however the HiSeq 2500 and HiSeq 2000 offer the best combination of speed, cost, and reagent kit compatibility.
  • Which Illumina sequencing instruments are compatible TruSeq Targeted RNA Expression?
    • TruSeq Targeted RNA Expression is currently supported on the MiSeq. MiSeq Reporter supports the protocol with its on-instrument analysis workflow.
  • Which Illumina sequencing platforms are compatible TruSeq RNA Access libraries?
    • TruSeq RNA Access Library Prep libraries are supported on all Illumina sequencing platforms.
  • Which Illumina-supplied consumables are necessary to run the GoldenGate Assay for Methylation?
    • GM-17-211 GoldenGate Methylation Cancer Panel I (standard panel oligo for methylation assay), GM-12-109 Sentrix Universal-96 Array Matrix (1,536-plex) , GM-95-201 Single-Use Activation Kit (576 Samples) or GM-95-202 Multiple-Use Activation Kit (576 Samples), GM-95-203 GoldenGate Assay Kit (96 Samples) or GM-95-204 GoldenGate Assay Kit (576 Samples), or GM-95-205 GoldenGate Assay Kit with UDG (96 Samples), or GM-95-206 GoldenGate Assay Kit with UDG (576 Samples)
  • Which Index Kit should I purchase if I will mainly be pooling less than 24 samples?
    • Illumina recommends purchasing the 24 Index/96 sample Kit (Catalog # FC-121-1011) if your sample number is not greater than 24 for each library prep event. Based on the possible index combinations, in order to optimize the indices in the kit, the 24 Index Kit is recommended.
  • Which MiSeq workflow should I choose when creating a sample sheet in Illumina Experiment Manager (IEM) for TruSeq ChIP samples?
    • For MiSeq runs, a sample sheet is required at the start of the run to enable analysis. You can choose either the "FASTQ Only" or the "ChIP-Seq" application in the "Other" category when creating a sample sheet in IEM. Both workflows will generate fastq files as data output.

  • Which SNPs would you recommend I use to design my GoldenGate OPA?
    • We strongly recommend using GoldenGate validated SNPs or two-hit validated SNPs with SNP scores (design scores) of 0.60 and above.
  • Which SNPs would you recommend I use to design my OPA?
    • We strongly recommend using SNPs from these two categories: GoldenGate validated SNPs and two-hit validated SNPs with SNP scores (design scores) of 0.60 and above. We discourage use of SNPs scoring < 0.40 as they have a low likelihood of converting into successful assays and they have the potential to adversely affect the performance of the entire OPA.
  • Which applications are supported on the MiSeq?
    • MiSeq Reporter, the secondary analysis software on the MiSeq, supports amplicon sequencing, targeted resequencing, small genome sequencing (de novo/resequencing), and small RNA applications. For complete information, see Sample Prep Applications for the MiSeq System.

  • Which assays and platforms are compatible with the PC Module?
    • The PC Module is compatible with GenomeStudio Genotyping projects created from Infinium and GoldenGate genotyping assays run on the iScan/HiScan, BeadXpress and BeadArray Reader.

  • Which flow cell should I use to perform an instrument wash on my HiSeq system?
    • Any previously used flow cell that has been stored properly in buffer can be used for the instrument wash. It is easiest to use the flow cell from the recently completed run to perform post-run and maintenance washes. Both v3 and v4 flow cells can be used to wash after high output modes.
  • Which flow cell should be used with Nextera Rapid Capture Enrichment libraries?
    • A paired-end flow cell from any Illumina sequencing system can be used with Nextera Rapid Capture Enrichment libraries.
  • Which genomic DNA storage buffer does Illumina recommend?
    • TE buffer with a composition of 10 mM Tris, pH7.5; 1 mM EDTA.
  • Which metrics can be used to evaluate data in the SNP Table to identify SNPs that require further editing and poorly performing SNPs?
    • SNPs can be evaluated and sorted by Call Freq, # no calls, Poly 10%, and Poly 50%.

  • Which metrics can be used to evaluate data in the Samples Table to identify poorly performing samples that should be excluded from the data?
    • Samples can be evaluated and sorted by Poly Call Rate, Poly 10%, and Poly 50%. We recommend using the scatterplot function within the Samples Table to plot Poly 50% against Poly Call Rate to graphically visualize sample outliers.

  • Which normalization method does Illumina recommend for miRNA data?
    • If technical replicates are used within a SAM or across multiple SAMs, Illumina recommends our newest Sample Scaling normalization, followed by Quantile normalization. If there are no technical replicates, we recommend Quantile normalization.
  • Which organisms are supported for TruSeq Targeted RNA Expression?
    • TruSeq Targeted RNA Expression is currently supported only for human, mouse, and rat.
  • Which parameters can be configured to manipulate the default clustering algorithms?
    • Minimum Number of Points in a Cluster, Cluster Distance, and Maximum Number of Clusters in the SNP Table, which can be found in the Clustering Options dialog box.

  • Which peaks should I use from the bioanalyzer for TruSeq Targeted RNA Expression?
    • Perform a region analysis on the bioanalyzer and define the region from 150–250 bases to get the molarity for the entire sample.
  • Which products are available for online downloads using the dmap Download Utiltiy?
    • Decode files for all BeadChip products except the HumanHap300 v.1.0 Genotyping BeadChip, Expression BeadChips ordered in 1- or 2-packs, iSelect Custom Genotyping BeadChips, and Sentrix Array Matrices (SAMs) are available using this software.
  • Which reagent position is used for the MiSeq flow check?
    • During the pre-run flow check, reagent is pulled from position 3 on the reagent cartridge, which is PR2.
  • Which reference genome version was used for designing Rapid Capture Exome and Expanded Exome Enrichment?
    • Human UCSC version hg19 which is the same as Genome Reference Consortium build 37 (GRCh37).
  • Which sequencing primers do I use for sequencing Nextera Mate Pair libraries: Nextera or TruSeq?
    • For sequencing purposes, the final completed Nextera Mate Pair libraries are technically considered to be TruSeq DNA libraries, and should be sequenced with TruSeq sequencing primers and workflows. The Nextera Mate Pair Kit actually employs both Nextera and TruSeq technologies. The genomic DNA is initially tagmented with a Nextera transposome enzyme, which adds a Mate Pair junction adapter to the ends of the DNA molecules. However, after circularization, the DNA is fragmented for a second time, at which point the TruSeq adapters are added. It is the TruSeq adapters that are utilized during the subsequent PCR, clustering and sequencing steps.
  • Which software should I use for downstream analysis of clusters and for generating genotype calls?
    • Illumina does not provide recommendations for downstream analysis outside of GenomeStudio.

  • Which species are available in DesignStudio for creating a Nextera Rapid Capture Custom Enrichment project?
    • The human genome (hg19 build) is currently available for custom enrichment projects.
  • Which systems are eligible for the HiSeq v4 upgrade?
    • System

      Serial Number

      HiSeq 2500

      SN# D00101 or higher

      HiSeq 1500

      SN# C00101 or higher

      HiSeq 2000

      SN# 7001403 or higher

      Field-upgraded HiSeq 2500

      SN# 7001403 or higher

      Field-upgraded HiSeq 1500

      SN# L179 or higher

      For more information, see the upgrade resources on the Overview tab.
  • Which version of OLB do I need to process my TruSeq data?
    • OLB 1.9 or higher must be used for processing control information; earlier versions of OLB will not be able to process controls.

  • Which version of Off-Line Basecaller (OLB) should I use to reprocess dual-indexed data from cif files?
    • Use OLB 1.9.3 or later to process data generated on HiSeq 2000, HiSeq 1000, or HiScanSQ. Use OLB 1.9.4 to process data generated on the Genome Analyzer.

  • Which workflow is required to create a TruSeq RNA Access Library Prep sample sheet using the Illumina Experiment Manager?
      • To create a HiSeq-compatible sample sheet, select the "HiSeq FASTQ Only" workflow and the "TruSeq LT" Sample Prep Kit.
      • To create a MiSeq-compatible sample sheet, select the "FASTQ Only" workflow and the "TruSeq LT" Sample Prep Kit.
  • Who can I call to get assistance in designing my NuPCR assays?
  • Why are SNPs included in the manifest of the Infinium HumanMethylation450 BeadChip?
    • SNPs were included on the Infinium HumanMethylation450 BeadChip so investigators could generate a DNA "fingerprint" of their samples as an added level of quality control. Please find further information in the Infinium HD Methylation Assay Protocol Guide. SNP assays on the BeadChip are not mentioned in the assay guide and only briefly described in the GenomeStudio Methylation Module. Follow this method to confirm the identity of samples from the same individual:

      • Highlight the SNP assays in the sample methylation profile, right-click, and "show only selected rows,"
      • For any given pair of samples that are supposed to be from the same individual, plot the beta values in a scatter plot from the sample methylation profile.
      • Samples from the same individual will look like this:

        Infinium HumanMethylation450 samples from one individual.

        Samples from different persons will look like this:

        Infinium HumanMethylation450 samples from different people.

        This is because the beta values calculated from SNP assays will cluster, much as they do in a standard genotyping theta graph. Samples from the same individual will have the SNP results fall along the identity line in a scatter plot, while samples from different persons will scatter into the nine different possible spots, based on their genotypes.

  • Why are some targets difficult to design in TruSeq Custom Amplicon's DesignStudio?
    • Homologs: if homologs are included in the same design, this can lead to low designability. We advise you to split homologs into separate CAT pools. GC Content: Note that regions with greater than 80% GC content can be difficult to design against, particularly if these regions are greater than 500bp in length. Homopolymer sequences and Repetitive elements: DesignStudio avoids these regions to ensure that probes have better specificity in the genome.
  • Why are the TruSeq Stranded Total RNA Sample Prep Ribo-Zero instructions different for Illumina protocols compared to Epicentre’s protocol?
    • Illumina has optimized the workflow to increase ease-of-use and scalability. Changes include the use of plates compatible with multi-channel pipettes, the streamlining of incubation steps, and an overall reduction in hands-on time.

  • Why are the polymerase and FFNs missing from the HiSeq SBS Kit v4?
    • The HiSeq v4 chemistry includes a pre-mixed incorporation reagent similar to the TruSeq Rapid kit.
  • Why are the sequencing intensity profiles so wobbly in the first 15 or so cycles in my run for my Nextera Rapid Capture Enrichment libraries?
    • This is a normal characteristic of Nextera tagmented libraries and relates to the sequence context at the tagmentation site.
  • Why can no custom reference be used? Is the reference generation itself proprietary? How easily can the process of creating a reference be implemented?
    • The Novel Indexing Scheme was pre-computed for the hg19 human reference and should be downloaded separately (67 GB size on hard drive).The reference is not proprietary. The open source version provides the tools to index any reference, however this require significant computational resource and is not supported by Illumina at the moment.
  • Why can't I find my ChIP-Seq data when I start a ChIP-Seq project in GenomeStudio?
    • The project creation wizard initially looks in the data repository directory, not the directories containing individual runs or projects, so you must point it at the correct directory. If there are sorted.txt files in multiple locations, such as \chip-data\run1\GERALD\sorted.txt and \chip-data\run2\GERALD\sorted.txt, then you should direct the wizard to \chip-data. It will search through any subfolders and display available runs.

  • Why can't I find the serial number of my BeadChips when I open the Decode File Download Utility?
    • Click Find to initiate a search. If there are still no chips visible, ensure that you requested data for a BeadChip that shipped at least 24 hours previously, and has not been on the server more than 30 days.
  • Why can't I successfully log in to the Decode File Download Utility?
    • You need a valid user ID and password for the Illumina iCom site and a working internet connection. Verify that your username and password are correct by logging in to your iCom account. If you do not have a login ID for iCom, complete and submit the form on the website for new users. If you believe you have a valid login ID and password for iCom, launch your browser and load any external page to verify you have a working internet connection. If you are still unable to log in, contact Technical Support .
  • Why do I have reads that end in adapter sequences in my small RNA sequencing results?
    • Reads longer than the small RNA molecule will sequence into the adapter. For this reason, we recommend sequencing for only enough cycles to cover the small RNAs of interest, or trimming the later cycles before aligning. The sequence that would need to be trimmed from the 3' end is given here for reference.

      TGGAATTCTCGGGTGCCAAGGAACTCCAGTCACnnnnnnATCTCGTATGCCGTCTTCTGCTTG

      This is the reverse complement of the index-specific primer sequence given in the Illumina Customer Sequence Letter, with the index sequence shown as nnnnnn.

  • Why do I have to do so many bead washings?
    • They are necessary to remove the rRNA.
  • Why do I have to log into my BaseSpace account to annotate variants in VariantStudio?
    • Illumina uses BaseSpace authentication to make sure that you are authorized to use the application. After you have authenticated for the first time, VariantStudio saves your credentials so that you are not asked to log in again later. You can delete your credentials using Annotation & Classification | Annotation Options | Forget BaseSpace Logon .
  • Why do I no longer get HGMD annotations when I annotate new variants in VariantStudio?
    • The annotation database used by VariantStudio software (both v1.0 and v2.1) to annotate variants loaded into the software no longer has HGMD information. This change means that neither version annotates newly loaded variants with variant-level HGMD information. With VariantStudio v2.1, Illumina has incorporated new disease annotation sources such as ClinVar, MedGen, GeneReviews, and SNOMED CT. In addition, to support cancer analysis and interpretation, information provided from the COSMIC database is expanded.

  • Why do my designed amplicons include SNPs in the probe regions when Avoid SNPs was turned on?
    • When Avoid SNPs is turned on, DesignStudio considers the location of polymorphisms and avoids them when possible. If this is not possible, it will place the probes in regions that do not interfere with their binding.
  • Why do some antibodies work for ChIP and others do not?
    • ChIP assays require that the epitope recognized by the antibody be available/exposed after cross-linking and not obscured in the middle of a protein complex. Antibodies have to be of very high avidity (strength of multiple interactions) so that the interaction with the protein will survive the washing steps. It also requires that the protein you are trying to immunoprecipitate cross-links efficiently to the chromatin.
  • Why does Eco Real-Time PCR system come in a 48-well format?
    • Our market research indicated that >70% of Real-Time PCR users run fewer than 50 wells per plate. In addition, the small well volume is optimized for a reaction volume between 5-20 µl, while 96-well plates are optimized for 25-50 µl reaction volumes. The 48-well plate enables you to save on the cost of reagents.

  • Why does Illumina recommend paired-end sequencing of stranded RNA libraries?
    • Due to the directional nature of the TruSeq Stranded RNA assays, paired-end sequencing captures the ends of the RNA molecule.

  • Why does the Nextera Rapid Capture Enrichment protocol require the TruTemp Heating System and MIDI plate insert?
    • The MIDI heat block used for tagmentation better supports automation and reduces the number of plate-to-plate sample transfers. The MIDI plate insert fits the 96-well, 0.8 ml "MIDI" plate used in the protocol.
  • Why does the Nextera Rapid Capture Enrichment protocol use a thermal cycler instead of a microheating system for the Rapid Capture hybridization reactions?
    • A thermal cycler leads to better consistency and more robust results across samples with a plate when compared to using a heat block for Rapid Capture hybridization.

      The lengthy incubation time needed at this step can lead to sample evaporation if the plate is not completely sealed. By using a thermal cycler at this step, the 100° heated lid helps to prevent large sample evaporation.

  • Why doesn't the Infinium HumanMethylation450 BeadChip contain specific distances from the CpG site to the transcriptional start site (TSS) of the listed genes?
    • The array density is much higher in the Infinium HumanMethylation450 BeadChip. There are a large number of CpGs for which multiple transcripts are listed and for which the CpG site can fall into different annotation categories. Consequently, the task of calculating TSS distances would be a large bioinformatic undertaking, and the numbers would have to be modified every time the genome was updated. Additionally, the column "UCSC_RefGene_Group" has content about the location of the CpG relative to specific regions and features of the associated genes, which is in many ways richer than the simple distance relative to transcriptional start site.
  • Why is GC high in the first few bases?
    • It is normal to observe both a slight GC bias and a distinctly non-random base composition over the first 12 bases of the data. For example, you might see it in the IVC (intensity versus cycle number) plots that are part of the output of Pipeline analysis software. In genomic DNA sequencing, the base composition is usually quite uniform across all bases, but in mRNA-Seq, the base composition is noticeably uneven across the first 10 to 12 bases. We believe this effect is caused by the "not so random" nature of the random priming process used in the protocol. This might explain why there is a slight overall G/C bias in the starting positions of each read. The first 12 bases probably represent the sites that were being primed by the hexamers used in the random priming process. The first 12 bases in the random priming full-length cDNA sequencing protocol (mRNA-seq) always have IVC plots that look like what has been described. This is because the random priming is not truly random and the first 12 bases (the length of two hexamers) are biased towards sequences that prime more efficiently. This is normal and expected.
  • Why is Illumina's Gene Expression assay called Direct Hyb?
    • Direct Hybridization (Direct Hyb) refers to the direct hybridization of probes on the array to the complementary cRNA molecule in your sample. This is in contrast to Illumina's flagship GoldenGate® Genotyping Assay, which incorporates a unique oligonucleotide onto the sample sequence. This unique oligonucleotide then hybridizes to the complementary probe on the array.

  • Why is a study number used for uploaded samples?
    •  A study number allows you to keep track of the samples you have uploaded, and provides an easy reference to identify your control samples for any publications you are preparing. It also allows others who are referring to your publications and studies to replicate the analysis by downloading the same samples.

  • Why is directional sequence information useful?
    • Directional sequence information is crucial in the identification of antisense transcripts for overlapping genes and it also increases the percentage of uniquely alignable reads in poorly-annotated species. Having directional information also eases the alignment and assembly processes for bioinformatics analyses.

  • Why is fragmentation buffer used instead of nebulization or sonication?
    • This fragmentation method takes advantage of the sensitivity of RNA to cleavage by divalent cations and temperature. This is the most robust method for fragmentation of RNA. Fragmentation by this method has been shown to result in more uniform sequencing coverage compared with other methods.
  • Why is overall intensity low?
    • This can be caused by a number of factors: Poor total-RNA quality. Check using an Agilent Bioanalyzer or Gel electrophoresis. Low factor setting. Increase the scanner factor setting. Expired or degraded reagents (e.g., Biotinylated UTP, Cy3, cleanup columns). Check the expiration dates on all reagents. Did not add the specified mass of cRNA to the array. Sample specific effects. Relatively few expressed genes (necrotic tissue). Stringency was too great in hybridization solution. Review the protocol for making up the hybridization solution. Temperature not set properly during hot-wash step. Review the protocol for making up the wash

  • Why is the message "Sequence Not Found" sent after I submit SNPs chosen using ADT?
    • This message means that the SNP sequence cannot be found in our internal database, which is a filtered version of dbSNP. We filter out any SNPs that are not appropriate for the Illumina platform, such as insertions/deletions, multiple nucleotide SNPs, and SNPs with ambiguous or multiple localizations.
  • Why is there a higher error rate for the first few bases?
    • The first two or three bases in mRNA-Seq reads have slightly elevated error rates compared to genomic DNA samples. We believe that this is an effect of the random priming process. The bases at the beginning of each read were likely at the back end of the random primer, away from the extending polymerase, during the priming process. It appears that this observation is a measurement of the mismatch pairing that is tolerated on the other end of the primer during the extension process by the polymerase.
  • Why is there an option for 'Tissue Source' in the query tool, but there is no Tissue Source for the samples?
    • At this time, uploading samples does not include this information. Future releases of GenomeStudio and BeadAccess will incorporate this ability, and at that time you will be able to query on this field.
  • Why is there more volume in my sample tube at the end of my rapid run than when I started?
    • At the end of all rapid sequencing runs, the instrument flushes water back into the sample tube to clean out the lines and prevent drying. This is an automatic procedure and does not require user intervention. This procedure dilutes sample left in the tube. Therefore, any sample left in the tube should be discarded after the run.
  • Why might I see different insert sizes when trying to aim for a 350 bp or 550 bp TruSeq Nano DNA Sample Prep insert size? How can I improve it?
    • Changing amounts of beads or volume inaccuracies during the beads size selection step will impact the final insert sizes. New users should take the TruSeq Sample Purification Bead Size Selection and Best Practices online training course prior to performing TruSeq Nano DNA sample preparation. The HS protocol may provide more consistency between samples.
  • Why must I gel purify before the PCR step?
    • To avoid amplification of adapter dimers.
  • Why were both Infinium I and Infinium II probes used for the Infinium HumanMethylation450 BeadChip? Will the design affect the data output / quality?
    • Assays were designed using Infinium II chemistry whenever possible for two reasons. First, these assays require only one beadtype per locus instead of two, saving space for other content. Second, Infinium II chemistry allows the use of degenerate oligonucleotides to design for every possible combination of methylation status across up to three CpG sites falling within a probe sequence (for a total of 23 possible combinations). For Infinium I assays, all underlying CpG sites were assumed to have the same methylation status as the query base. 

  • Will CASAVA 1.8.2 be able to process both single-index and dual-index TruSeq runs?
    • Yes. Specific input commands to CASAVA 1.8.2 will determine whether the data are demultiplexed as single-index or dual-index. However, single and dual indexes cannot be combined in the same CASAVA command. Separate instances of CASAVA are required to process single-index and dual-index runs (i.e.: run CASAVA twice) and use 2 different sample sheets, and demultiplex the TruSeq HT lanes separately from the TruSeq LT/v2 lanes. Different use base masks will be required to demultiplex different lanes. Please see the CASAVA User Guide for more information.

  • Will I need to re-validate HCS 1.5/RTA 1.13 if upgrading from HCS 1.4/RTA 1.12?
    • Validation of HCS 1.5/RTA 1.13 should not be required if you are upgrading from HCS 1.4/RTA 1.12. Changes in RTA do not affect data quality, and changes in HCS updated the user interface to enable dual indexing. Please refer to the HCS 1.5 Release Notes for additional information about new features in this software package.
  • Will ROX reference dye interfere with NuPCR reactions?
    • If your experimental protocol uses ROX as a passive reference, you should not design assays with ROX dye.