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Categories

Assays
  1. Chromatin Immunoprecipitation Sequencing (ChIP-Seq)
  2. Direct Hybridization Assay
  3. GoldenGate Genotyping Assay
  4. GoldenGate Methylation Assay
  5. Infinium DNA Analysis Assay
  6. Infinium Methylation Assay
  7. miRNA Expression Profiling Assay
  8. mRNA-Seq
  9. Multiplex Sequencing
  10. Nextera Sample Prep
  11. Paired-End Sequencing
  12. Single-Read Sequencing
  13. Small RNA Discovery and Analysis
  14. TruSeq Sample Prep
  15. VeraCode GoldenGate Genotyping Assay
  16. VeraCode GoldenGate Methylation Assay
  17. VeraCode Protein Assay
  18. Whole-Genome DASL HT Assay
Epigenetics
  1. Array-Based Methylation Analysis
  2. ChIP-Seq
  3. Sequencing-Based Methylation Analysis
  4. Small RNA Analysis
Software
  1. Assay Design Tool (ADT)
  2. BaseSpace
  3. CASAVA
  4. Decode File Client
  5. DesignStudio
  6. Eco Control Software (ECS)
  7. Experiment Manager
  8. GenomeStudio
  9. HiSeq Control Software (HCS)
  10. iControlDB
  11. KaryoStudio
  12. MiSeq Control Software (MCS)
  13. MiSeq Reporter
  14. OLB
  15. RTA
  16. Sequencer System Software
  17. Sequencing Analysis Viewer (SAV)
  18. Sequencing Control Software (SCS)
Systems
  1. BeadXpress Reader
  2. cBot
  3. Cluster Station
  4. Eco Real-Time PCR System
  5. Genome Analyzer IIe
  6. Genome Analyzer IIx
  7. HiScanSQ
  8. HiSeq 1000
  9. HiSeq 1500
  10. HiSeq 2000
  11. HiSeq 2500
  12. MiSeq
Technologies
  1. General Questions
  2. Illumina Array
  3. Illumina Sequencing
  4. Real-Time PCR
Workflow
  1. Analysis Software
  2. Experimental Design
  3. General Information
  4. Instrumentation
  5. Product and Support Files
  6. Sample Preparation
  • 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 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 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 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 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 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 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 other sequencing flow cells compatible with MiSeq?

    No. The MiSeq uses a flow cell that is specifically designed for the MiSeq.

  • 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 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 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 there any in-line controls in the sample prep?
    No.
  • 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 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 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 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 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 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 -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.
  • Are TruSeq Cluster Kits v3 and SBS Kits v3 available for use on the Genome Analyzer?

    No. These kits are for use on HiSeq and HiScanSQ only.

  • 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 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.
  • 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 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 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 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 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 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 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 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 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 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 cifs with BaseSpace?
    No. Even if you do choose to save cifs, they will be saved only locally and will not be transferred to BaseSpace.
  • Can I analyze Rapid run data with Casava?
    Yes, you can analyze Rapid run data with Casava. Please note that 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 perform alignment of each flow cell’s data 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 combine my SBS kits to achieve read lengths longer than what is provided in a kit?
    Yes. Please refer to the “Preparing SBS Reagents” section of the HiSeq 2500 User Guide or HiSeq 1500 User Guide for instructions and supported sequencing read lengths.
  • 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 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 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 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 generate a call report?
    A call report can only be generated from the GenomeStudio Genotyping Module.
  • Can I get bead-level data?
    Yes, bead-level data is available. Please contact Technical Support for assistance with this feature.
  • 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 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 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 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 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 perform a primer rehyb on the MiSeq?
    No, a primer rehyb is not possible on the MiSeq.
  • 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 process my sample with low sequence diversity?

    Yes, there are two ways to compensate for low sequence diversity so that matrix and phasing intensity calibration parameters can be accurately estimated.

    1. Mix in phix control library at 30% - 50% with your sample to increase base diversity.

    2. Perform template hybridization on the cBot using the TruSeq Rapid Duo cBot Sample Loading Kit (Cat# CT-402-4001). Load a balanced sample in one lane and your low diversity sample in the second lane. Then when setting up run parameters on the HiSeq system, be sure to set the balanced sample as the Control lane.

  • 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 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 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 GenomeStudio software on a VMware virtual machine?
    Yes. However, this is not officially supported by Illumina due to very slow performance.
  • 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 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 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 cifs with HCS 2.x?
    Yes. HCS 2.x enables you to save cifs.
  • 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 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 split my 200-cycle kit for shorter runs?
    Yes. You can split the 200-cycle SBS Kit into two equal volumes suitable for up to 101 cycles each. See the TruSeq SBS Kit Reagent Preparation Guide (200 Cycles) for instructions and storage requirements. If you need smaller volumes for shorter runs, Illumina recommends using the TruSeq SBS Kit (50 Cycles).
  • 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 (flow cell) of my HiSeq 2500 system?
    Yes. If you have just completed two Rapid flow cell runs on side A and B and would like to set up a High Output flow cell run on side A, switch modes on side A only. Similarly, you must switch modes on side B to perform a High Output flow cell run on side B. Perform mode change procedures on both sides of the system concurrently to save time and obtain the greatest run flexibility.
  • 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 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 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 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 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 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 analyse TruSeq Custom Amplicon and TruSeq Ampicon Cancer Panel data from a HiSeq system?
    No, file directory structures are incompatible with MiSeq Reporter software.
  • 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 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 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 same reagents on the HiSeq that I use on my Genome Analyzer?
    No. Cluster Kits and SBS Kits for the HiSeq are not equivalent or compatible with the Genome Analyzer. Likewise, Cluster Kits and SBS Kits for the Genome Analyzer are not equivalent or compatible with the HiSeq.
  • 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 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 Nextera DNA Sample Preparation kits be used in TruSeq Exome Enrichment/Custom Enrichment?
    This is not supported at this time.
  • 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 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 SBS reagents and High Output SBS reagents be combined together?
    No. Do not combine Rapid SBS reagents and High Output SBS reagents. Each set of reagents has been formulated specifically for use with only its respective flow cell and instrument run parameters.
  • 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 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 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 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 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 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 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 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 TruSeq Exome Enrichment Kits be used for methylation applications?
    The TruSeq Exome Enrichment kit protocol is not currently compatible with bisulfite sequencing.
  • 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 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.
  • cnvPartition did not find the exact breakpoint of my found region. Is there a way to adjust this?
    Yes, you can adjust the start and stop positions of a found region by right-clicking the found region and adjusting the genomic positions. You may need to use the zooming and panning functions to determine the exact position to which you would like to adjust the found region.
  • 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 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 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 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 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 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 the new bcl2fastq software package?
    You will need bcl2fastq to handle zipped bcl files. It can also handle regular (non zipped) bcl files.
  • 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 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 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 Rapid Run mode?
    No. Priming is integrated into the HCS workflow for Rapid Runs.
  • 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 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 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 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 flow cell as for a High Output flow cell?
    Cluster density tends to be about 15% higher on Rapid 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 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 protocols available for whole-genome or reduced-representation bisulfite sequencing?
    Yes, we have protocols available for whole-genome bisulfite sequencing (methylC-Seq), as well as reduced representation bisulfite sequencing. Note that these are Illumina-demonstrated protocols and are offered with associated restrictions and conditions. Protocols for other methods are available through third-party publications. Examples are included on our website.
  • 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. It must be in Casava format, and all samples listed in BaseSpace must be indexed or all must not be indexed (for example, you cannot have a mix of indexed and unindexed samples in a single flow cell when using BaseSpace).
  • 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 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 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 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 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 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 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 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 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 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 HiSeq require an uninterruptible power supply (UPS)?
    Illumina provides a region-specific UPS with the HiSeq instrument.
  • 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 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 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 following files are uploaded as instrument health data: 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/
  • 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?
    See the GenomeStudio RNA Sequencing Module User Guide in the GenomeStudio Portal for more information.
  • 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 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 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 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 obtain KaryoStudio?
    Contact your account manager for information about getting new versions of KaryoStudio software.
  • How can I obtain new versions of cnvPartition?
    Updated versions can be obtained through the GenomeStudio Portal or by visiting http://www.illumina.com/iconnect.
  • 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 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 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 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 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 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 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?

    You can specify the GenerateFASTQ workflow in your sample sheet, which creates FASTQ files and then exits secondary analysis. For more information, see the MiSeq Sample Sheet Quick Reference Guide.

  • 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. Some widely used 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/). 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 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 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 for BaseSpace and Rapid runs?

    All BaseSpace runs and any runs that will be demultiplexed with bcl2fastq or Casava will 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 should include information for just two lanes, as opposed to the eight lanes included in a regular, High Output run sample sheet. You can create sample sheets for Rapid runs manually, using Excel or a text editor, or you can created them by manually editing Casava sample sheets generated with IEM.

    If you are using BaseSpace for data storage and analysis, a sample sheet will be required for both Rapid runs and High Output runs. If you are using BaseSpace for run monitoring only, a sample sheet is not needed.

    If you are analyzing data locally, Rapid runs can be converted with bcl2fastq without using a sample sheet; in this case the results will be 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 the majority of cases, throttling of the BaseSpace Broker data upload should not be necessary. Throttling may be needed if you need to apply greater control over network bandwidth usage (such as at sites at which instruments share the network with other users or sites with limited upload speed). Throttling may be necessary in certain scenarios, such as when local network connectivity is temporarily lost and then restored, causing BaseSpace Broker to suddenly consume more network bandwidth as it tries to “catch up” with the upload of accumulated data. In these types of scenarios, if no throttling is applied, BaseSpace Broker could consume all available bandwidth on the network until the backlog of data is cleared. If throttling is to be applied and if the local network permits, Illumina suggests throttling to higher than the 10Mbit/sec minimum specification. 20Mbit/s (approximately 3Mbyte/s = 24Mbit/s) is recommended. This will provide BaseSpace Broker with enough bandwidth to recover in the case of data upload delays.

    If you need to throttle BaseSpace Broker, please supply the following instructions to your local IT administrator:

    Throttling of BaseSpace is done on the HiSeq-PC by application rather than by IP address.

    1. In windows, open the Local Policy Editor by opening a cmd window and running gpedit.msc.

    2. Expand the Computer Configuration / Windows Settings nodes.

    3. Select Policy-based QoS.

    4. Right-click and then choose Create new policy. Then, perform the following steps:

    a. Enter a name.

    b. Uncheck Specify DSCP value.

    c. Select Specify Outbound Throttle Rate, and then enter 3 Mbytes/s (or 24Mbit/s). This should enable the system to catch-up if necessary.

    d. Select Next.

    e. Select Only applications with this executable name, and then enter Illumina.BaseSpace.Broker.exe.

    f. Click Next (this policy applies to any source IP and target IP addresses).

    g. Click Finish (this policy applies to all ports and protocols).

  • 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 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 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 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 merge data from two HiSeq flow cells?

    With Casava, you can merge data from different HiSeq flow cells (different runs) with Casava 1.8.2’s configureBuild script.

    First, align the data (samples) from each flow cell with Casava 1.8.2 separately, using configureAlignment. Then, include each Sample Directory as an input directory in the configureBuild.pl command line. You can specify input directories by using the –id option, as detailed on page 100 of the Casava 1.8.2 User Guide Rev C.

    With BaseSpace, a sample merge application is available, which enables you to merge data for a single sample that originated from different flow cells. Merging is performed prior to alignment analysis of the sample 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 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?
    Please refer to the Designing Custom GoldenGate Assays technical note.
  • 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 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 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 HiSeq run data to BaseSpace?

    HiSeq Run data can be uploaded to BaseSpace only during a sequencing run and only if you have selected the BaseSpace option in HCS. Please see the HiSeq User Guide for information on setting up a run for upload to BaseSpace.

    For more information on BaseSpace, or to set up a free BaseSpace account, please go to 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 use BaseSpace for Run Monitoring only (for example, for SAV functionality in BaseSpace)?
    You must select run monitoring on the Storage tab of HCS during Run Configuration.
  • How do I use BaseSpace for run monitoring?
    The BaseSpace run monitoring option enables you to monitor an ongoing run’s progress remotely by logging into your BaseSpace account. You will need to select the Run Monitoring option during run setup. Then, you can log into your BaseSpace account from anywhere and view your run in the BaseSpace version of Sequence Analysis Viewer.
  • 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 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 the HCS 2.x data compression options change data analysis or data handling?

    If a run has zipped bcls, you will need to use a new software tool, Bcl2Fastq, 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 zipped or non-zipped bcl files.

    If you send your data to BaseSpace, bcl to fastq conversion and demultiplexing will be performed automatically following the completion of the data upload.

  • 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 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 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 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 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 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 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 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 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 instrument health upload option communicated to customers?
    The first time the HCS 2.x (or later) 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 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 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 flow cells be stored?
    After amplification, linearization, blocking, and primer hybridization, you can store the flow cell in the provided storage at 2° to 8°C for up to ten days.
  • How long can Rapid SBS reagents be stored after preparation
    Prepare Rapid SBS reagents the night before or on the day of use. If prepared the night before, you can store reagents in their original bottle with the cap tightened. If prepared the same day, set reagents on ice at 4°C until you are ready to load them onto the instrument.
  • 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 Rapid water wash and maintenance wash?
    A Rapid water wash takes approximately 15 minutes while a Rapid maintenance wash takes approximately 35 minutes. Please refer to HiSeq 1500/2500 Mode and Instrument Wash for additional details relating to instrument washes.
  • How long does it take to complete a run on the MiSeq?

    Generally, a run takes between 4 hours and approximately 55 hours depending on the number of cycles you perform. See the MiSeq System Product Information Sheet for complete information.

  • 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 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 switch between Modes on my HiSeq system?
    It takes about 45 minutes to change from High Output mode to Rapid mode and about four hours to change from Rapid mode to High Output mode. The time spent for mode switching is in addition to the instrument wash performed at the end of each run. For more information, see the HiSeq 1500 and 2500 Mode and Instrument Washing Insert. See the HiSeq 1500 and 2500 Mode and Instrument Washing Insert for details.
  • 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 cBot take to perform the template hybridization procedure on a 2 lane flow cell?
    Just under one hour.
  • How long is each Index Read?
    Both Index 1 (i7) and Index 2 (i5) are 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 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 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 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 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 of sequencing reagents are in my TruSeq SBS kits?

    TruSeq Rapid SBS: 200 cycle kit contains reagents for 225 total cycles, and the 50 cycle kit contains reagents for 74 total cycles.

    TruSeq v3 SBS kit volumes (High Output runs) have not been changed. The 200 cycle kit contains reagents for 209 total cycles, and the 50 cycle kit contains reagents for 58 total cycles.

  • 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 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 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 does the MiSeq flow cell have?

    The MiSeq flow cell is a single-lane flow cell.

  • 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 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 I load into KaryoStudio?

    There currently is no limit on the number of samples that can be loaded into KaryoStudio.

    Refer to Appendix A of the KaryoStudio Software User Guide or see the KaryoStudio Benchmark Performance Technical Note for more information about KaryoStudio software performance.

  • 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 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 swaths and tiles on a Rapid two-lane flow cell?
    Scanning and analysis of a Rapid two-lane 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 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 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 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 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 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 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 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 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 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?
    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 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 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 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 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 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.

  • 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 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 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 use BaseSpace for HiSeq Run Monitoring only, what files are sent to BaseSpace?
    The HiSeq will send only the InterOp folder, RunInfo.xml file, and RunParameters.xml file.
  • 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.
  • 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 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 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 DNA fragmentation (i.e., with Covaris) necessary?
    No. The tagmentation step in the Nextera protocol eliminates the need for mechanical fragmentation/shearing.
  • 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 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 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 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 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 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 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 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 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 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 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 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 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 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 1.8.2?
    Yes. bcl2fastq is used in place of the configureBcl2fastq step in Casava. The output of bcl2fastq is fastq.gz files organized into Project and sample directories as specified in the sample sheet. This output will be compatible with the configureAlignment and configureBuild components of Casava 1.8.2. The sample sheet format required for bcl2fastq is equivalent to Casava 1.8.2 sample sheet format, and it is described in the Bcl Conversion with bcl2fastq User Guide.
  • 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 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 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 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 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 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 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 LIMS support for Infinium Methylation BeadChips?

    LIMS support is available for the Infinium HumanMethyation450 BeadChip.

  • 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 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.

  • 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.
  • 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.
  • 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.
  • 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 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 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.

  • 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 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 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 minimum hardware recommendations to run GenomeStudio software?

    The table below includes Illumina’s minimum hardware recommendations to run GenomeStudio software.

    ParameterSequencing Data AnalysisMicroarray Data AnalysisMicroarray and Sequencing Data Analysis
    CPU SpeedIntel Pentium 2.0 GHz or fasterIntel Celeron Duo or fasterIntel Celeron Duo or faster
    Processor64-bit64-bit64-bit
    Memory Size4 GB or more8 GB or more8 GB or more
    Hard Drive250 GB or larger100 GB or larger250 GB or larger
    Video Display1,280 x 1,0241,280 x 1,0241,280 x 1,024
    Operating SystemWindows XP, Vista, or Windows 7Windows XP, Vista, or Windows 7Windows XP, Vista, or Windows 7
    Specific OS RequirementsMicrosoft .NET Framework 3.5Microsoft .NET Framework 3.5Microsoft .NET Framework 3.5
    Network Connection1 GbE or faster1 GbE or faster1 GbE or faster
  • 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 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 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 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 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 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 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 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 “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 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 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 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 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 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 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 network requirements for data transfer from the HiSeq to a server?
    You need a one gigabit connection per instrument between the instrument computer and the server. For more information, see the HiSeq System Site Preparation Guide.
  • 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 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 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 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 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 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 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 data compression options are available for HiSeq runs using HCS 2.x?

    HCS 2.x features the two data compression options: bcl file zipping and Qscores binning. These options are available during run setup in HCS 2.x. If you are using BaseSpace for data storage and analysis, bcl files will be zipped by default. If you are using BaseSpace for only run monitoring, bcl zipping is not required. Other run folder files are unchanged from previous versions of HCS.

    If you are not using BaseSpace, the data compression options are as follows:
    1. No compression
    2. Zip bcls + bin Qscores
    3. Zip bcls only

    If you are using BaseSpace, the data compression options are as follows:
    1. Zip bcls only (required for BaseSpace)
    2. Zip bcls + bin Qscores

  • 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 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 do ‘TOP/BOT’ strand and ‘A/B’ alleles mean in the Illumina manifests and reports?
    Please refer to our technical note entitled “TOP/BOT” Strand and “A/B” Allele (PDF).
  • 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 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 do to make sure my HiSeq is ready to send data to BaseSpace?
    If you want to upload data from a HiSeq to BaseSpace, you will need a minimum upstream connection bandwidth of 10Mbit/second per instrument. You can assess network speed using free online tools, such as www.speedtest.net.
  • 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 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 '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 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 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 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 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 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 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 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 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 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 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 my SNP is not in your database?
    You can submit the SNP in a SequenceList.
  • 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 Illumina sequencing platforms are compatible with Nextera?
    Nextera is compatible with all Illumina sequencing platforms: MiSeq , HiSeq 2000, HiSeq 1000, HiScanSQ , and GAIIx.
  • 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 "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 "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 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 matrix file?
    The matrix file is used for base calling and accounts for cross talk between dyes.
  • 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 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 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 bcl2fastq?
    bcl2fastq is a separate piece of stand-alone software that is run on a Linux scientific computing system. You can get the installer from MyIllumina. Dependencies are equivalent to those for Casava 1.8.2 and are outlined in the bcl2fastq user guide. They are also listed on pages 114 to 117 of the Casava 1.8.2 User Guide Rev C. If the option to compress bcl files was selected during run setup, then conversion of bcl files will require the use of the bcl2fastq.
  • What is deconvolution?
    It is the ability to distinguish between two or more clusters that are in close proximity to each other.
  • 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 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 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 instrument health data, and where can I find more information about the option to send instrument health data to Illumina?
    HCS software allows HiSeq instruments connected to the internet to send instrument health information to Illumina. This information will be anonymous and will include only generic run metrics. This information will be used by Illumina to help improve Illumina’s products. If you wish to turn off this option, or would like further information, please go to the Options menu in the HiSeq control software. You can find the Options menu under Menu|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 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 acquisition time for the Eco system?

    The acquisition time for all four channels is approximately six seconds.

  • What is the alternative SBS workflow?

    TruSeq SBS v3 reagents enable an alternative workflow for loading all SBS reagents at the start of a 2x101-cycle sequencing run for both Read 1 and Read 2. Using this workflow might result in a slight increase in phasing in Read 2, which should not result in a decrease in quality.

  • What is the amount of sequencing required on the various Illumina sequencers?
    The amount of coverage depends on the size of the sample genome and the type of sample. For more information, see the Coverage Calculation Tech Note and the Optimizing Coverage for Targeted Resequencing Tech Note.
  • 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 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 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 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 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 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 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 expected correlation for hybridization replicates between BeadChips? Between arrays within a BeadChip?

    r2 between BeadChips: .99

    r2 within BeadChip: .99

  • 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 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 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 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 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 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 input material for small RNA sequencing?
    The protocol was optimized using 1.0 μg of total RNA.
  • 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 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 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 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 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 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 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 temperature resolution of the Eco system?

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

  • 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 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 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 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 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 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 operating system is used on the instrument computer?
    The HiSeq instrument computer employs 64-bit Windows Vista.
  • What operating system is used on the MiSeq?

    The MiSeq runs on Windows 7 Embedded Standard.

  • 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?
    Please contact your local IT administrator if local security policies will need to be modified to allow access to BaseSpace. BaseSpace uses SSL/https port 443 and the domains api.basespace.illumina.com and basespace.illumina.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 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 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 starting material?
    Double Stranded DNA specific dyes, such as QuBit or picogreen.
  • What reagents do I need to perform bisulfite sequencing applications?

    A complete list of consumables and equipment required to perform bisulfite sequencing applications can be found in the Whole-Genome Bisulfite Sequencing (WGBS) and Reduced Representation Bisulfite Sequencing (RRBS) protocols. A list of compatible kits can be found on the epigenetics section of the Illumina website .  

  • 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 sample prep do I use for MiSeq?

    A comprehensive list of easy and rapid sample prep options can be found on the MiSeq kits page. To determine which sample method is best suited for your application, see Sample Prep Applications for the MiSeq System.

  • 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 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 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 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 total-RNA quantification method do you recommend?
    Any routine method is fine. Illumina recommends using RiboGreen to quantify total-RNA concentration.
  • 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 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 SBS kits are available for the HiSeq?
    Illumina currently offers two SBS kits for the HiSeq: a 200-cycle kit and a 50-cycle kit. Both kits contain the same formulations and only differ in volume.
  • What version USB is on the MiSeq?
    The MiSeq is equipped with USB 2.0.
  • What versions of instruments, reagents, and software are compatible?

    Go to the sequencing Version Compatiblity Reference at www.illumina.com/VersionCompatibility.

  • 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 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’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 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 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 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 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 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 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 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 HCS data compression options?
    Please see the Illumina whitepaper, Reducing Whole-Genome Data Storage Footprint, which is available on MyIllumina. You can find it on the HiSeq Documentation and Literature support pages.
  • 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 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 do I load my samples on the cBot if I am using the TruSeq Rapid Duo cBot Sample loading kit?
    Load 135 ul 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 eight-port and 10-port gaskets with my Rapid two-lane and High Output eight-lane flow cell, respectively?
    For setting up both Rapid and High Output flow cells, load the eight-port gasket in the back manifold position, and load the 10-port gasket in the front manifold position.
  • 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 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 washes on my HiSeq system?
    Any previously used sequencing flow cell that has been stored properly in buffer can be used for system washes. It is easiest to use the flow cell from a recently completed sequencing run for performing post-run and maintenance washes.
  • Which genomic DNA storage buffer does Illumina recommend?
    TE buffer with a composition of 10 mM Tris, pH7.5; 1 mM EDTA.
  • 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 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 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 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 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 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 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 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.

  • 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 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 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 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 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 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 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 my SBS v3 reagent waste brown?

    Due to an interaction with one of the v3 reagents, SRE, waste appears dark brown in color and has a stronger odor. This is normal. The change in waste color does not impact performance and is not toxic. You might see a discoloration on the funnel caps and SRE sipper line. Any spills will be dark brown in color as well.

  • 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 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 cBot plates be updated with the sequencing primers required for dual-indexed Nextera libraries and TruSeq HT libraries?

    Not at this time. If you are sequencing Nextera libraries, you need sequencing primers provided in the TruSeq Dual Index Sequencing Primer Box for any run types. If you are sequencing dual-indexed TruSeq HT libraries on a single-read flow cell, you need sequencing primers provided in the TruSeq Dual Index Sequencing Primer Box, Single Read.

  • Will I have enough reagents in my 200-Cycle SBS Kit to perform a 2x100 cycle run with indexing?
    Yes. The TruSeq SBS Kit (200 Cycles) contains sufficient reagents for 209 cycles of sequencing, which covers 101 cycles for Read 1, 7 cycles for the Index Read, and 101 cycles for Read 2.
  • 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 local proxies affect BaseSpace?
    No testing has been done on the effects of local proxies on BaseSpace access.
  • Will my data be kept confidential?
    Yes. All data generated by us will be kept strictly confidential. The data belongs solely to the submitting investigator or company. Illumina will only accept anonymized samples with no patient association.
  • Will the new Nextera dual-indexing support single-indexing as well?
    Yes. 12-plex single-index runs are supported on the HiSeq/GA using i7 indexes only. GA support for Nextera will be available in November 2011 when the latest version of SCS/RTA is available. Please see the Nextera DNA Sample Preparation Guide for more information on pooling recommendations for samples less than the full kit size.
  • Would there be issues demultiplexing current TruSeq libraries if a dual index run was done?
    No. You must specify the correct –use-bases-mask and use the appropriate sample sheet to enable demultiplexing. Please see the CASAVA User Guide for more details on sample sheets and demultiplexing commands.