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Can I make libraries from RNA with DV200 < 30%?
Success is not guaranteed with poor quality libraries. For libraries with a DV200 < 30%, input of 100 ng or greater is recommended.
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.
For some library prep, AMPure XP beads are user-supplied from Beckman-Coulter Genomics. See the appropriate TruSeq DNA or RNA library prep guide for more information.
All beads required for the protocol are supplied in the kit.
Yes, the TruSeq Amplicon - Cancer Panel has been optimized for use with DNA derived from FFPE sources. The input amount depends on the ∆Cq. For more information, see the TruSeq FFPE DNA Library Prep QC Reference Guide (part # 15067391).
We recommend FFPE-derived DNA only be used with TruSeq Custom Amplicon Low Input when using short amplicon lengths of 150 or 175 bp. Shorter amplicons provide better amplification than longer ones when the sample input is fragmented FFPE-derived DNA. For running TruSeq Custom Amplicon Low Input prep with FFPE samples, you must use the Illumina TruSeq FFPE DNA Library Prep QC Kit to determine input amount based on ∆Cq.
Only use FFPE-derived DNA with TruSeq Custom Amplicon when using short amplicon lengths of 150 or 175 bp. Shorter amplicons provide better amplification than longer ones when the sample input is fragmented FFPE-derived DNA. To run TruSeq Custom Amplicon prep with FFPE samples, use the Illumina TruSeq FFPE DNA Library Prep QC Kit to determine input amount based on ∆Cq.
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.
All NextSeq kits include reagents for paired-end sequencing and a paired-end flow cell. However, single-read runs are possible on a paired-end flow cell when the run is set up as a single-read run.
Nextera Rapid Capture Enrichment index 1 (i7) and index 2 (i5) sequences are identical to index sequences used in other Nextera based kits. They use the Read 1 (HP10), Read 2 (HP11) and Index 1 (HP12) sequencing primers.
Although index sequences are identical across Nextera kits, the index 2 primer reagents (i5, E501-E502) are not interchangeable across kits. Also, the Nextera Rapid Capture Enrichment kits use only two index 2 (i5, E501-E502) sequences compared to eight index 2 (i5) sequences used in other Nextera kits.
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.
No. These kits are for use on HiSeq and HiScanSQ only.
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.
All validated Illumina library prep methods are supported to run on the HiSeq. However, make sure that you match samples with the desired output to maximize workflow efficiency.
TruSeq DNA PCR-Free and TruSeq Nano DNA LT library prep kit adapters are methylated. However, TruSeq HT library prep kit adapters are not methylated and are therefore not suitable for bisulfite sequencing applications.
TruSeq Rapid Exome Index 1 (i7) and Index 2 (i5) sequences are identical to index sequences used in other Nextera based kits. They use the Read 1 (HP10), Read 2 (HP11), and Index 1 (HP12) sequencing primers. Although index sequences are identical across Nextera kits, the index 2 primer reagents (i5, E505-E506) are not interchangeable across kits. Also, the TruSeq Rapid Exome kits use only 2 Index 2 (i5, E505-E506) sequences compared to 8 Index 2 (i5) sequences used in other Nextera kits.
Yes, this kit targets 160 bp of the 5' and 3' UTR of every targeted gene.
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.
Yes, there are examples of data from HiSeq sequencing runs in BaseSpace.
The quality of DNA isolated from FFPE samples can be highly variable. Due to this variability, it is difficult to predict the quality of a library prepared from FFPE samples using the TruSight Cardio protocol. Illumina does not support FFPE or degraded DNA as input. FFPE samples can be attempted using the TruSight Cardio protocol, but failed libraries originating from this sample type are not eligible for replacement or troubleshooting by Illumina.
The quality of DNA isolated from FFPE samples can be highly variable. Due to this variability, it is difficult to predict the quality of a library prepared from FFPE samples using the TruSight One protocol. Illumina does not support FFPE or degraded DNA as input. FFPE samples can be attempted using the TruSight One protocol, but failed libraries originating from this sample type are not eligible for replacement or troubleshooting by Illumina.
The quality of DNA isolated from FFPE samples can be highly variable. Due to this variability, it is very difficult to reliably predict the quality of a library prepared from FFPE samples using this protocol. Illumina does not support FFPE or degraded DNA as input for this this protocol. This does not mean that FFPE samples cannot be attempted, but that failed libraries originating from this sample type are not eligible for replacement or troubleshooting by Illumina.
Yes, these libraries can be used on all HiSeq, MiSeq, HiScanSQ and GA instruments.
Yes, VariantStudio v2.1projects are compatible with VariantStudio v2.2. Samples, annotations, including any imported custom annotations, and filter favorites are all carried over to VariantStudio v2.2.
No, original manifolds do not align correctly on the new instrument.
No. The MiSeq uses a flow cell that is specifically designed for the MiSeq.
Ideally, perform pre-PCR and post-PCR procedures in 2 separate rooms. If separate equipment is used (e.g., shaker, heat block, centrifuge) and a strict cleaning schedule is maintained, then the assay can be performed in designated, nonadjacent areas in the same room. Regular bleaching is outlined in the reference guide. In addition, filtered pipette tips are also recommended.
Illumina adapter sequences are provided in the Illumina Customer Sequence Letter.
The adapter plate is not compatible with TruSeq RNA v1 or v2 kits as the protocol and the reagents are different.
Yes, they are different. See the Downloads page on the kit support page.
The same controls are used, but at different concentrations.
Eco does not use PMTs or photodiodes, it uses a CCD camera as the detector which is designed to compensate for dark current.
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.
Yes, download the BED files from Downloads page.
The TruSeq Nano DNA Library Prep Kit uses the same workflow as TruSeq DNA Sample Prep Kit. No changes have been made in analysis.
The TruSeq ChIP Sample Prep kit does not contain in-line controls due to the low input amount.
VariantStudio requires the installation of .NET Framework 4.0 (Full, not 4.0 Client Profile). If .NET 4.0 is not detected, the installer requests that you install the framework. Another prerequisite for installation is the use of a 64-bit version of Windows.
You need to increase your starting template concentration. However, the starting template volume (120 µl) is unchanged from previous kits.
Yes. The number of PCR cycles has been optimized for various assay conditions taking into account both the number of amplicons per reaction and the type and amount of input DNA (eg FFPE). The recommended number of PCR cycles will ensure sufficient yield for the bead-based normalization while avoiding overamplification.
No. The HiSeq X Reagent Kit v2.5 has the same workflow introduced with the HiSeq X Reagent Kit v2. The HiSeq X Reagent Kit v2.5 uses the same library denaturation step during reagent preparation before clustering on the cBot. The denaturation step coupled with enhanced exclusion amplification (ExAmp) reagents enables sequencing of TruSeq DNA PCR-Free libraries on the HiSeq X system, in addition to superior data quality and coverage.
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.
Yes. Using the NextSeq 500/550 Kit v2 requires NextSeq Control Software (NCS) v1.4.
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.
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.
No, the cBot plates are the same; however, if sequencing TruSeq HT libraries on single-read flow cells, the TruSeq Dual Index Sequencing Primer Box, Single Read (single use box) (catalog# FC-121-1003) is necessary in order to sequence both index reads.
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.
There are no in-line controls for the TruSeq Small RNA Library Prep Kit.
There are no in-line controls for the small RNA kit.
Based on the amplicon coverage and binding sites of the long-range PCR primers, the following ambiguities are known to be present in TruSight HLA results. Common and Well-Documented (CWD)1 alleles are highlighted in bold.
1 Mack SJ, Cano P, Hollenbach JA et al. Common and well-documented HLA alleles: 2012 update to the CWD catalogue. Tissue Antigens 2013: 81: 194-203.
2 Voorter CE, Lardy NM, van den Ber-Loonen EM. Presence of the DRB4*0103102N null allele in different DRB1*04-positive individuals. Tissue Antigens 2000: 55(1): 37-43.
This list is based on testing over 500 samples with broad coverage of ethnicity and allele combinations.
There are 3 amplicon ambiguities found using the IMGT/HLA 3.23 database with TruSight HLA v2.
DRB1*12:01:01 and DRB1*12:10 are ambiguous due to the forward primer location. DRB1*12:01:01 and DRB1*12:10 are both part of the DRB1*12:01:01 G group. They are distinguished from each other in a single base position in Exon 1 (IMGT Codon -16, exon 1 base position 40). The DRB1*12:10 codon -16 is ATT (Ile) and the DRB1*12:01 codon is GTT (Val). The TruSight HLA v2 DRB1 amplicon begins in intron 1 and does not cover exon 1. Coverage of exon 1 was excluded from the assay because it would require a 14 kb amplicon that would be challenging to amplify reliably.
DPB1*13:01:01 and DPB1*107:01 are ambiguous for similar reasons to the ambiguity previously described. DPB1*13:01:01 and DPB1*107:01 are both part of the DPB1*13:01:01 G group. The TruSight HLA v2 amplicon does not cover exon 1. These alleles are distinguished from each other at 2 base positions in exon 1(IMGT Codon -22, base position 24 and Codon -14, base position 47). Codon -22 is GCG (Ala) in DPB1*13:01:01 and GCA (Ala) in DPB1*107:01. Codon -14 is ACG (Thr) in DPB1*13:01:01 and ATG (Met) in DPB1*107:01. Coverage of exon 1 was excluded from the assay because it would require an amplicon that would be challenging to amplify reliable.
DRB1*08:01:01 and DRB1*08:01:03 are ambiguous in IMGT/HLA database version 3.23. As of March 2016 and the release of IMGT/HLA 3.24, 08:01:03 allele has been removed from the database because “sequence shown to contain errors and be identical to DRB1*08:01:01.”
In addition to these amplicon ambiguities, conditional ambiguities arise in DPB1 and DQB1. We define a conditional ambiguity as an ambiguity present only when 2 alleles are paired, but may not be present when 1 or both alleles are paired with other alleles. These conditional ambiguities appear in DPB1 because of lack of polymorphic sites in intron 2. For example, DPB1*04:01 paired with a DPB1*04:02 is ambiguous with DPB1*105:01 and DPB1*126:01 due to loss of phase across intron 2 as the gap between heterozygous positions is too great to phase. However, DPB1*04:01 paired with DPB1*16:01 is unambiguous for both alleles.
See the TruSeq Library Prep Pooling Guide (part # 15042173) for low plexity index guidelines. Pooling guidelines are only required when performing TruSeq library prep for sequencing on the following systems:
There are no fixed guidelines for variant filtering in VariantStudio. Appropriate filter settings depend on the intended use of the assay and must be determined for each project. See the VariantStudio User Guide for more information on the filtering options.
Assign 1.0 Project Files (*.cgp) are not compatible with Assign 2.0. However, Assign 2.0 can import and analyze FASTQ files generated by TruSight HLA Sequencing Panel (version 1).
Yes, but only after validating the software per institution, local, state, and federal guidelines before using the Illumina BaseSpace Variant Interpreter software.
The protocol has been optimized for fragments of 150 bp insert size. Altering this length could cause sample loss during bead size selection steps or inefficient probe binding.
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.
No. HiSeq v2 and TruSeq rapid SBS reagents cannot be combined. Each set of reagents has been formulated specifically for use only with its respective flow cell and instrument run parameters. The cluster kits and flow cells are also paired with respective kit types and cannot be used interchangeably.
No. HiSeq v4 and TruSeq v3 high output SBS reagents cannot be combined. Each set of reagents has been formulated specifically for use only with its respective flow cell and instrument run parameters. The cluster kits and flow cells are also paired with respective kit types and cannot be used interchangeably.
The TruSeq Amplicon - Cancer Panel is a presynthesized, fixed content panel. As such, the content cannot be modified. However, the genomic targets from the cancer panel can be used as a starting point for a customized TruSeq Custom Amplicon design using DesignStudio.
No, there is no range of hybridization times. The protocol has been optimized for 30 minutes for each hybridization
Currently the ramp rate is not adjustable. Check back periodically and keep an eye on the monthly Illuminotes newsletter for updates.
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.
No, .cif files cannot be analyzed with BaseSpace. Additionally, it is not possible to output .cif files with HCS v2.2 on HiSeq v4 mode or Rapid Run mode with HiSeq v2 chemistry. The option to output .cif files is available in TruSeq v3 mode and Rapid Run mode with TruSeq chemistry.
If you are using CASAVA, you can analyze rapid run data with CASAVA. If the zip BCL files option was chosen during run set up, you will need to use the bcl2fastq converter in place of the configureBclToFastq component of CASAVA. For rapid runs, you will align data from each flow cell separately and then merge the data at the configureBuild step.
If you are not using CASAVA, note that Illumina is discontinuing distribution of CASAVA software to better support new products available on BaseSpace. BaseSpace features analysis options for a large array of NGS applications.
If you currently use CASAVA, you can analyze HiSeq v4 data with CASAVA. You need to use the bcl2fastq v1.8.4 conversion software in place of the configureBclToFastq component of CASAVA.
For HiSeq v4 runs, perform alignment of data from each separately, and then merge the data at the configureBuild step.
If you are not using CASAVA, note that Illumina is discontinuing distribution of CASAVA software to better support new products available on BaseSpace. BaseSpace features analysis options for a large array of NGS applications.
No, this is not an option in the PC Module.
Yes, when adjusting the analysis settings from a current analysis, a new analysis is initiated. Additionally, the alteration of sample indexes or sample types automatically initiates a new analysis.
Some options can be modified and the options vary by assay. For more information, see the appropriate library prep reference guide.
You can cluster a GA flow cell on the cBot 2, but sample tracking is available only for clustering a HiSeq flow cell (excepting TruSeq Rapid flow cells). To perform cluster generation on a GA flow cell, you must turn off sample tracking and change the adapter plate. Dedicating a cBot 2 to 1 platform avoids routinely reconfiguring the instrument for sample tracking. For instructions on changing the adapater plate, see the cBot 2 System Guide (document # 15065681).
Yes, but make sure that the library type you cluster on the cBot 2 is compatible with your sequencing instrument. For example, HiSeq X is compatible with TruSeq Nano DNA and TruSeq DNA PCR-Free libraries only.
As with any molecular biology techniques, a change in protocol will give a change in absolute results. We do not recommend comparing absolute counts (or counts normalized to total counts) between different preparation protocols. However, comparison of fold change between the two protocols should yield good correlation.
The data is comparable to TruSeq RNA Sample Prep v2 data. The TruSeq stranded RNA and Directional mRNA-Seq protocols are based on distinct ligation chemistries and the libraries they produce are expected to differ as a result. Direct sample to sample comparison is not recommended for these library types.
Yes, if the data from different runs is normalized with same normalizer genes. The raw data from different runs cannot be merged together into a single dataset. For more information, see the MiSeq Reporter User Guide.
NCS v1.4 is not compatible with the NextSeq 500 Kit (v1). Use NCS v1.3, or earlier, with v1 kits.
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.
Yes, sequences can be edited using the Navigator functionality. Edits are flagged in the Coverage View and can be found using the forward and reverse arrows in the Navigator. There is an option to include sequence edits on the report, which shows the edit, base position, and user.
Yes. Direct sample to sample comparison can be performed using the ForenSeq Universal Analysis software.
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.
There are variances between the TruSeq LT and HT kits that may result in differences in yield and the amount of adapter dimer for the final library. Be sure to take extra care pipetting the AMPure XP beads accurately in the Clean Up PCR step, to minimize the amount of adapter dimer carried through to the final library. Also, note that the AMPure XP bead ratio at this step is optimized for the longer length of the dual-index adapters.
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.
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.
Sample consensus sequences can be exported from Assign 2.0 in MS Excel (*.xls, *.xlsx, or *.xlsm depending on MS Excel version), text (*.txt), or FASTA (*.fasta) formats.
Yes, you can export the consensus FASTQ sequences for each sample and each locus.
Yes, the adapter plate can undergo freeze/thawing up to 4 times. More freeze thaws of the plates may impair assay performance.
It is possible to use larger fragment sizes, however, we have found that shorter fragment sizes generate the best coverage. Please refer to Appendix A in the user guide for the TruSeq RNA sample prep kit that you are using for more information.
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.
Yes. VariantStudio v2.2 and v2.2 can be installed concurrently on the same computer. The two versions of VariantStudio operate independently; information is saved in different application spaces. VariantStudio v2.1 installs as "Illumina VariantStudio 2.1", while v2.1 installs as "Illumina VariantStudio 2.2". Therefore, VariantStudio v2.2 does not overwrite the v2.1 installation. Using the default installation folder for both versions guarantees concurrent usability. If you decide to define a custom installation path, make sure that you do not use the same installation folder for both versions.
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.
Yes, the software accepts VCF files produced by the Genome Analysis Toolkit (GATK) v1.6.
Yes. However, if analysis software other than Illumina analysis software is used to generate data, the VCF file might not contain the columns required by VariantStudio. See the VariantStudio User Guide (part # 15040890 B) for information about VCF file requirements.
Yes. You can import custom annotations to the software using the Custom Annotation function in VariantStudio. This feature requires that a custom annotation file uses a tab-delimited text format (TSV) and contains a header row that specifies the column names. VariantStudio allows both custom variant annotations and custom gene annotations.
Yes. You can import custom annotations in tab-delimited text (*.txt) file format into the software from the Custom Annotations tab in Settings. For more information, see the BaseSpace Variant Interpreter Beta Online Help.
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.
Yes. Metadata can be added to a sample at any point, with options to add it when importing the VCF files (recommended). After importing VCF files, sample metadata can still be added or edited using the software interface or a sample metadata sheet. However it is added, metadata autopopulates the appropriate section of the report.
Assign can be installed on a network drive. For network assistance, consult your facility IT administrator.
Assign 2.0 can be installed on a network drive, server, or individual PC. In addition, multiple types of installations can be installed in the same facility.
No. The software uses the following default classification schemes for tumor and germline samples:
Yes. By default, VariantStudio provides five classification categories: Pathogenic, Presumed Pathogenic, Unknown Significance, Likely Benign, and Benign. You can edit these categories through the Annotation & Classification | Classification Settings menu.
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.
Yes, the SNP list is available for download:
Yes, please use the following catalog numbers:
FT-260-1002 for Infinium OncoArray FastTrack Service
FT-260-1012 for Infinium OncoArray+ FastTrack Service (for the OncoArray plus add-on)
Yes. Please select the appropriate workflow based on the flow cell type (Single Read or Paired End) when starting your run so that the correct chemistry is used. If using a single-read flow cell the TruSeq Dual Index Sequencing Primer Box, Single Read (single use box) (catalog# FC-121-1003) is necessary in order to sequence both index reads.
No. Comparisons of two or more groups of samples cannot be performed using the application.
No. BaseSpace Variant Interpreter does not support comparisons of 2 or more groups of samples. Use BaseSpace Cohort Analyzer for cohort or population analysis.
BaseSpace Variant Interpreter supports family-based analysis for singletons (proband only), duo (proband plus one parent), trios (proband plus both parents), and extended pedigree (proband, both parents, and up to 5 siblings). For more information, see the BaseSpace Variant Interpreter Beta Online Help.
VariantStudio supports family-based filtering of father, mother, affected child, and affected or unaffected siblings. In VariantStudio v2.2, the analysis requires input of the proband and one other sample, either a parent or a sibling. This process filters variants that are consistent with a particular inheritance mode, including autosomal dominant, autosomal recessive, X-linked recessive, and de novo mutation.
Performing 2 x 76 bp runs is recommended for optimal performance with fusion callers. If the paired reads are overlapping, fusion calling can be less efficient.
Yes. Sample to sample comparison is done through the Cross Sample Subtraction Filter. This process filters variants that are present in one sample but not the other.
No, BaseSpace Variant Interpreter does not currently support direct comparison of analysis results from 2 different samples.
Yes, TruSight HLA v2 uses the Nextera XT Indexes. 384 unique Nextera XT index combinations are available, so index barcode cycling for runs of fewer than 384 samples is easily achievable.
Up to 12 tagmented samples can be pooled and processed together for the enrichment part of the protocol. However, pooling is not performed with the 8 rxn × 1 plex kit.
No, load the library card when it is on the stage to avoid spilling oil or disturbing the contents after loading.
The HiSeq 3000 system has been tested and validated to process a broad array of samples from whole exome, whole genome, and RNA sequencing. Libraries with low complexity within the first 25 bp, such as single amplicon or 16S samples, are not expected to produce quality results.
The HiSeq 4000 system has been tested and validated to process a broad array of samples from whole exome, whole genome, and RNA sequencing. Libraries with low complexity within the first 25 bp, such as single amplicon or 16S samples, are not expected to produce quality results.
Real-Time Analysis (RTA) v1.18 includes optimizations to the algorithms that identify clusters and estimate the color normalization matrix and phasing and prephasing rates. These optimizations improve the ability of Real-Time Analysis to handle low-diversity samples, such as samples with unbalanced genome compositions (AT- or GC-rich genomes) or samples with low sequence diversity (amplicon sequencing). Because of these improvements, it is no longer necessary to designate a control lane in the control software to estimate matrix and phasing. For details, see Low-Diversity Sequencing on the Illumina HiSeq Platform.
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 MethylationEPIC Array using an FFPE Methylation Protocol (available in 2016) along with the Infinium FFPE DNA Restoration Solution kit.
To run FFPE samples on the InfiniumMethylationEPIC Array, perform the following procedures:
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.
Illumina does not support, and strongly advises against, running libraries prepared by different sample prep kits in the same lane of a flow cell. We do support running libraries prepared by different sample prep kits in different lanes of the same flow cell or spiking in llumina PhiX control library in the same lane as any user-prepared libraries. If different library types are run in different lanes, Dual Index Recipes and the Dual Index Primer Box must be used. The indices between TruSeq HT and Nextera are unique and not shared. Please see appropriate sample prep user guides for index sequences.
Yes. However, for optimal coverage, Illumina recommends sequencing stranded RNA libraries with paired-end chemistry.
Yes, use the Cufflinks Assembly & DE BaseSpace App.
Yes. You can run any library on the MiSeq provided your library has complementary adapters.
No, only runs of the same mode can be performed simultaneously. If you run TruSeq v3 mode on side A, then you must run TruSeq v3 mode on side B. The same is true for running HiSeq v4 mode. For Rapid Run mode, you can perform a rapid run on both sides using TruSeq Rapid kits on one side and HiSeq Rapid v2 kits on the other.
In HiSeq v4 mode .cif files cannot be saved. The option to save .cif files is available in other modes.
Yes. After selecting filter settings using options in the Filters pane, you can save the filter for later use. To apply a saved filter, click the drop-down menu in the Current field, and select a filter name from the list. The filter is automatically applied.
Yes. Any filter combination can be saved and applied to other data sets. For more information, see the BaseSpace Variant Interpreter Beta Online Help.
Yes. All Nextera-based libraries use the same sequencing and index primers (HP10, HP11, HP12) and can be loaded in different lanes of the same flow cell for sequencing.
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:
MiSeq Reporter will generate the standard Resequencing reports. Consequently, you will need to zoom in on your enriched regions to obtain relevant data.
No, the BeadChip and flow cell fit in the same imaging compartment stage and use the same cameras.
Yes; however, do not store the BeadChip adapter without a BeadChip on the imaging compartment stage on the NextSeq 550 system.
A single side of the instrument can be switched from one mode to the other. However, after a run begins on one side, a run cannot be started on the other side unless it is the same mode. Perform mode switching procedures on the side that you intend to sequence on in the new mode. For example, if you have completed 2 rapid runs on side A and B, and want to set up only 1 high output flow cell on side A, change the mode for only side A.
A high output run on side B cannot be performed until a mode change is complete on side B. For efficiency and the most run flexibility, perform mode switching on both sides of the system at the same time.
No. An upgrade package (catalog # SY-401-4002) is available for HiSeq 3000 only.
No. The cBot 2 was specifically designed to provide positive sample tracking.
Assign 2.0 software was developed to work with data generated using TruSight HLA kits and Illumina instruments. Other sequencing kits and platforms have not been tested and are not supported. Assign 2.0 is compatible with FASTQ files generated from both the TruSight HLA Sequencing Panel (version 1) and TruSight HLA v2 Sequencing Panel.
The Assign software was specifically developed to work with data generated using TruSight HLA kits and Illumina MiSeq instruments. Other sequencing kits and platforms have not been tested and are not supported.
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.
No. HiSeq X flow cells are designed and optimized for use on the HiSeq X system. HiSeq X flow cells are not compatible with other HiSeq models.
No. HiSeq X reagents are designed and optimized for HiSeq X systems. HiSeq X reagents are not compatible with other HiSeq models.
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.
No. The Illumina VariantStudio End User License Agreement (EULA) states that Illumina VariantStudio must be used solely to analyze data generated from an Illumina sequencing instrument.
No. File directory structures from a HiSeq system are incompatible with MiSeq Reporter software.
However, the TruSeq Amplicon App is available in BaseSpace and can be used to analyze the TruSeq Amplicon - Cancer Panel, TruSeq Custom Amplicon Panel, and the TruSight Myeloid Sequencing Panel.
No, file directory structures are incompatible with MiSeq Reporter software. However, the TruSeq Amplicon App is available in BaseSpace and can be used to analyze the TruSeq Amplicon Cancer Panel, the TruSight Myeloid Sequencing Panel, and the TruSeq Custom Amplicon panels.
No. The TruSeq Rapid Exome Enrichment reagents and protocol have been optimized to be used together. The kits include all components for both library prep and Rapid Capture. Each kit contains components optimized specifically for the use in that protocol. Deviation from the protocol with reagents from other kits can lead to protocol underperformance or failure.
Yes, NuPCR works on all instruments from major manufacturers. It has been shown to work on the following instruments:
Yes. SAV 1.8.46 or later is required to view data from a HiSeq 3000 run. You can download SAV for use on a computer or use BaseSpace.
For the version of SAV compatible with your version of HCS, see the HCS release notes.
Yes. SAV 1.8.46 or later is required to view data from a HiSeq 4000 run. You can download SAV for use on a computer or use BaseSpace.
For the version of SAV compatible with your version of HCS, see the HCS release notes.
Yes. SAV 1.8.37, or later, is required to view data from a HiSeq X run. You can download SAV for use on a networked computer or use SAV in BaseSpace.
Yes. Use SAV 1.8.36, or later to view sequencing data from a NextSeq system. You can download SAV for use on a networked computer or use SAV in BaseSpace. SAV 1.8.36 is also compatible with HiSeq and MiSeq run data.
The kit includes a sufficient volume of Sample Purification Beads reagent to process the intended number of samples for the kit. Do not use beads or columns from any other manufacturer with this protocol.
No, kits are not interchangeable.
Yes, the low sample (LS) and high sample (HS) workflows may be applied with both the high throughput (HT) and low throughput (LT) kit configurations. Please refer to the user guide for the TruSeq library prep kit that you are using for Illumina's recommendations on kit and protocol combinations to ensure the best results.
The release of NCS v1.4 and the NextSeq 500/550 Kit v2 together enable the use of a custom Index 2 primer. Using a custom Index 2 primer is not possible with previous kit or control software versions.
This protocol has been optimized and validated using the microheating system specified in the library prep reference guide. Comparable performance is not guaranteed when using alternate equipment.
Illumina does not recommend reusing plates as there is a significant risk of contamination.
A sample sheet can be loaded during the Load Library Card step. If you are not using the default index adapter layout, create and edit a sample sheet to record information about your samples and indexes. Illumina recommends that you use IEM to create an appropriate sample sheet. For more information, see the Illumina Experiment Manager User Guide and IEM NeoPrep quick reference card.
Illumina recommends using a microheating system during tagmentation and enrichment wash steps. However, if a microheating system is not available, a thermal cycler can be used. Set up the Tagmentation reaction as instructed in the User Guide using a 96-well hard-shell plate. For the enrichment wash steps, the large sample volume requires adjustments to be made to the protocol for robust performance. Refer to Appendix A in the User Guide for complete details.
No. The cBot 2 manifold includes clearances to allow the flow cell scanner to read the flow cell barcode. The new manifolds replace the existing manifolds in all cBot cluster kits.
Use the NextSeq 500/550 Kit v2 with NCS v1.4.
The NextSeq 500 Kit (v1) is not compatible with NCS v1.4. If using the NextSeq 500 Kit (v1), do not upgrade your control software beyond NCS v1.3.
Yes. All cluster kits intended for use on the cBot are also compatible with cBot 2.
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.
No. The same sequencing primer is distributed across both lanes of a rapid flow cell as part of the HiSeq 2500 on-instrument cluster generation workflow.
Do not use adapters other than those provided in NeoPrep library prep kits. The adapters are especially formulated to work in the digital microfluidics environment.
Yes, up to 500 ng of input has been tested.
The new HiSeq v4 reagent kits now support dual indexing workflows without requiring the purchase of additional SBS agents. Sample prep for dual-indexed libraries requires that both indexes 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 8-base single-indexed sequencing run.
For libraries, you can use unlabeled 8-tube strips on the cBot 2 only for runs without sample tracking. Use an unlableled 8-tube strip for all custom or additional primers, regardless of run type.
All TruSeq DNA sample prep kits, including the HT version, supports input of standard genomic DNA only. Illumina offers other kits for Amplicon library prep. The TruSeq DNA HT Sample Prep kit supports high quality genomic DNA as input for sample prep to ensure best results. Please see the DNA Input Recommendations section of the TruSeq DNA Sample Preparation Guide for further information.
The adapter plate is single-use per well and Illumina recommends that it not undergo more than four freeze/thaw cycles. For this reason, Illumina strongly recommends processing at least 24 samples at a time with the TruSeq HT library can be pooled and sequenced in any Illumina supported configuration including low-plex. If it is desired to prepare only a few samples at a time, Illumina recommends purchasing a TruSeq LT library prep kit.
Yes, the modified cycling incubation can be used on the GoldenGate Methylation Assay. It is not required, however, because that assay is more tolerant towards incomplete conversion.
Yes. Third-party analysis options require sequencing data in the FASTQ file format. Use the Local Run Manager Generate FASTQ analysis module to convert data for later use. The Generate FASTQ analysis module converts base calls to FASTQ files and then exits the workflow. No further analysis is performed.
Yes, as long as the index read primer and multiplex read 2 primer are used. TruSeq DNA and RNA libraries have the same architecture and sequencing primer attachment sites as v2 multiplexed libraries.
Illumina does not support running libraries prepared by different sample prep kits in the same lane of a flow cell and does recommend running any combination of library types in a single lane. Please note that Illumina PhiX can be spiked in to any lane with user-prepared libraries.
No, not currently. The Directional mRNA-Seq Sample Preparation protocol would need to be optimized to take the longer adapters into account, as the size difference makes adapter dimers more difficult to distinguish from actual inserts using SPRI or column purifications. Illumina encourages customers to continue to use the Illumina Small RNA v1.5 adapters as specified in the Directional mRNA-Seq Sample Preparation Guide.
TruSeq adapters are methylated. However, the kits are not currently compatible for the following reasons:
Sequence TruSight HLA libraries using paired-end 250 base pair (2 x 250 bp) reads. Only the HiSeq 2500 in rapid run mode supports 2 x 250 bp read length. However, TruSight HLA libraries have not been extensively tested on the HiSeq 2500.
Sequence TruSight HLA libraries using paired-end 250 base pair (2 x 250 bp) reads. The NextSeq system currently supports a maximum of 2 x 150 bp sequencing. Testing has shown a significant decrease in HLA typing accuracy and an increase in ambiguity rate with these shorter read lengths. For best results, sequence TruSight HLA libraries on the MiSeq system.
Yes, the TruSight One Sequencing Panel is designed to allow trio sequencing and data analysis using the MiSeq system. The 9-sample TruSight One kit is intended for use with the MiSeq and contains three MiSeq v3 reagent cartridges. The 36-sample kit does not contain sequencing reagents and can be sequenced on any Illumina sequencer.
To log in to MiSeq FGx Control Software, a connection must be established with the ForenSeq Universal Analysis Software server to check authentication. After authentication, an RUO run can be performed without connection to the server. There is a buffering mechanism with the MiSeq FGx Control Software to allow data transmission to resume upon a connection loss to ForenSeq Universal Analsyis Software during acquisition.
Yes, the nucleotide distance of all the indices is such that mismatch of 1 still makes a unique index.
Illumina recommends that you purchase and install an anti-virus software of your choice to protect the instrument control computer against viruses. For more information, see the NeoPrep Library Prep System Site Preparation Guide.
Custom primers have not been tested for use on the HiSeq 3000.
Custom primers have not been tested for use on the HiSeq 4000.
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).
Yes. There are three reservoirs on the NextSeq reagent cartridge reserved for custom primers: #7 for a custom Read 1 primer, #8 for a custom Read 2 primer, and #9 for a custom Index 1 primer or custom Index 2 primer.
Using a custom Index 2 primer requires the NextSeq 500/550 Kit v2 and NCS v1.4. Using a custom Index 2 primer is not possible with previous kit or control software versions.
Degraded DNA generates libraries with the kit. Depending on the extent of degradation, not all of the large amplicons in the multiplex can be detected. The kit can also overcome high levels of inhibitors that can be present in forensic samples.
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.
Only Covaris shearing is supported.
No, a polyploid project (.pcm) can only be opened using the PC Module.
For paired-end flow cells, only Index 1 can be re-hybed, and this must occur before Index 2 begins. For single-read flow cells, Index 1 and Index 2 can be rehybridized before the run completes.
The protocol is optimized for the inclusion of a gel size selection step selecting for a DNA insert of 150–200 bp. Longer inserts could be used, however, this may decrease the signal to noise for the binding motif and may require modification for optimal run performance. Illumina recommends selecting a gel size range of 250–300 bp for the final library.
If pooling fewer than the number of indices provided in the kit, it is necessary to consider low-plexity index combinations. Color balance during the index read is needed to ensure proper image registration. If there is no signal in one of the color channels (red or green) of the index read, image registration may fail and no base will be called for that cycle. If no base is called, the index read may not be able to be matched to the sequence specified in the sample sheet, and then samples will not be able to be demultiplexed. Refer to the TruSeq Library Prep Pooling Guide for recommendations and guidelines for Illumina sequencing systems that require balanced index combinations. The Illumina Experiment Manager (IEM) will give warnings when generating sample sheets if the index combinations do not meet this requirement.
Assign 2.0 can be installed on a shared server or network drive, and multiple users can independently launch and simultaneously use the same instance of Assign 2.0. These instances share only the settings file and the data under review can be from the same sample. However, edits, comments, and user-defined settings are not available between users.
Yes. Report templates created and saved in VariantStudio are available to other VariantStudio projects.
Coverage of GC regions can be impacted by the model, settings, and performance of the thermal cycler used. Illumina has validated the Bio-Rad DNA Engine Tetrad 2, the Bio-Rad S1000, and the MJ Research PTC-225 DNA Engine Tetrad thermal cyclers. Other thermal cyclers may differ in their performance across the genome.
Yes, TruSeq cluster kits are backwards-compatible.
No. Rapid run SBS reagents and high output SBS reagents cannot be combined. Each set of reagents has been formulated specifically for use only with its respective flow cell and instrument run parameters. The cluster kits and flow cells are also paired with respective kit types and cannot be combined.
Yes, samples can be analyzed in a single experiment. We have observed high concordance between detected probes using MCS3 versus MCS4 plus RTE and have found the variability in raw signal intensity correlations between MCS3 and MCS4 plus RTE to be comparable to the variability between freshly made MCS3 and MCS3 nearing the end of its shelf life. As is to be expected, signal intensity correlations and detected probe concordance decrease with input RNA quality.
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.
No, there is currently no Small RNA v1.5 oligo-only kit available.
Illumina has not validated the array for 5-hMc. However, publications have used the Infinium HumanMethylation450K array for 5-hMc analysis and it is possible that this protocol will works on the Infinium MethylationEPIC array.
For more information, see Nazor, Kristopher L., et al. "Application of a low cost array-based technique—TAB-Array—for quantifying and mapping both 5mC and 5hmC at single base resolution in human pluripotent stem cells." Genomics 104.5 (2014): 358-367.
Yes. The MiSeq FGx Control Software offers an RUO run option alongside the Forensic Genomics run options. The RUO side of the software is fully compatible with all Illumina-supported RUO run types, and runs are set up as for standard MiSeq runs. For more details on set-up and workflow, see the MiSeq FGx Instrument Reference Guide.
Yes, the same metrics that are shown during data acquisition are available on the ForenSeq Universal Analysis Software after the run is complete.
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.
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.
The DSN Normalization protocol was optimized for pre-TruSeq libraries. The TruSeq RNA libraries are not readily amenable to the DSN treatment. There are two primary problems:
Yes. To run the protocol without the controls, substitute Resuspension Buffer (RSB) for the control mixture at each step. See the appropriate TruSeq DNA or RNA sample preparation guide for details.
Illumina does not recommend using the microTUBE AFA Fiber Screw-Cap tube (Covaris Part # 520096) with the TruSeq Nano DNA Library Prep protocol. The solution tends to stick to the sides of the tubes and to the two rods inside the tube, resulting with probable sample loss. Illumina has optimized the M220 shearing conditions using the microTUBE AFA Fiber Pre-Slit Snap-Cap (Covaris Part # 520045) tube and the microTUBE AFA Fiber Crimp-Cap tubes (Covaris Part # 520052).
The protocol is optimized for 18 cycles of PCR to allow for variability in sample input amounts and quality. High quality samples will likely be able to use a lower number of PCR cycles and still generated adequate amounts of final library. A titration of PCR cycles can be performed to identify the optimal performance for particular samples. Illumina does not recommending increasing the number of PCR cycles as it may skew the representation of the library.
No. However, a sample sheet created using IEM is compatible with the RUO mode of the MiSeq FGx Control Software. For forensic runs, the samples are defined in the ForenSeq Universal Analysis Software before acquisition through a file upload or manual entry. For details on the information in the file, see the ForenSeq Universal Analsyis Software Guide.
No, the wells cannot be reused.
The MiSeq provides sufficient output to run a single sample per MiSeq run. However, the HiSeq 2500 is the most cost effective and highest throughput platform for exome sequencing.
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.
No, variant classification and associated notes saved in the Classification Database are not versioned. If the classification category or notes are modified for a given variant in the Classification Database, previous information is overwritten with the current modification.
Currently, batch processing of multiple Eco systems is not supported. Check back periodically and keep an eye on the monthly Illuminotes newsletter for updates.
On average, there are several hundred long-read molecules per well. Individual wells produce many long read fragments randomly sampled from across the genome.
Yes, in order for the results to be transferred from the MiSeq FGx to the ForenSeq Universal Analysis Software a connection must exist between the systems. In many cases that connection is established through the company network. However, in some labs that connection can be on a local network created with the provided router.
A blue dye is used in NeoPrep library prep reagents to aid in loading and collection. It appears as a characteristic peak at 200–250 bp in a BioAnalyzer trace and is not indicative of issues with the final library. The blue dye has no impact on downstream sequencing.
Fragment Analyzer does not show the blue dye peak in the trace due to dilution on that instrument.
Yes, the MiSeq FGx needs must be washed between runs to remove any remaining reagents from the fluidics lines and make sure that they are clean before the next run is started. The MiSeq FGx offers a bleach post-run wash protocol that minimizes run-to-run contamination. See the MiSeq FGx Instrument Reference Guide for wash instructions.
Yes, a cBot is needed for cluster generation on any 8-lane high output flow cell, including HiSeq v4 and TruSeq v3.
Yes, a cBot is needed for cluster generation on any 8-lane high output flow cell, including the HiSeq 3000/4000 patterned flow cell. On-instrument clustering is a feature of Rapid Run mode, which is available only on the HiSeq 2500 and HiSeq 1500. You can use any cBot running cBot software v3.0 or later to cluster a HiSeq 3000/4000.
Yes, a cBot is needed for cluster generation on any 8-lane high output flow cell, including the HiSeq 3000/4000 patterned flow cell. On-instrument clustering is a feature of Rapid Run mode, which is available only on the HiSeq 2500 and HiSeq 1500. You can use any cBot running cBot software v3.0 or later to cluster a HiSeq 3000/4000.
You can transfer data from a sequencing run to BaseSpace for analysis and storage. Additionally, you can configure the NextSeq system to transfer data to a local server and perform analysis using third-party software.
The NeoPrep System has a touch screen monitor with on-screen keyboard to enable on-instrument configuration and set-up using the software interface. A mouse and/or keyboard can be connected via the external USB ports if preferred. The system can also be customized to disable the on-screen keyboard and only use an external keyboard, however, these are not provided with the system.
In general no, because data from a run can be saved locally. However, the following operations require an internet connection:
Yes. To convert zipped .bcl files, use bcl2fastq v1.8.4. This version can also convert non-zipped .bcl files.
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.
No, only 1 of the gene fusion partners needs to be detected. The enrichment approach allows you to pull down the target and the partner fusion gene with it.
If sequencing TruSeq HT libraries with the HiSeq, HiScanSQ, or GAIIx system on single-read flow cells, the TruSeq Dual Index Sequencing Primer Box, Single Read (single use box) (catalog # FC-121-1003) will need to be ordered separately when using both indices. This add-on box is not required if sequencing a TruSeq HT prepared library with the MiSeq System or on paired-end flow cells for HiSeq, HiScanSQ, GAIIx. Additional SBS reagents may be necessary to account for all index cycles.
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.
No. BaseSpace Variant Interpreter is a stand-alone, software as a service (SaaS), cloud-based solution. Although it integrates with your BaseSpace Sequence Hub account, you can create a separate BaseSpace Variant Interpreter account. If you have a MyIllumina account, you can use those credentials to log on to both applications.
Yes. Priming SBS reagents is required. The control software prompts for this step, which takes about 15 minutes.
No. Priming is integrated into the HCS workflow for Rapid Run mode.
No, TruSeq DNA PCR-Free library prep kits contain Sample Purification Beads (SPB) that are used for size selection and clean-up steps. They do not require the user to supply AMPure beads separately.
No, the kit contains Sample Purification Beads (SPB) that are used for size selection and clean-up steps. They do not require the user to supply AMPure beads separately.
The TruSeq Dual Index Sequencing Primer Kit (catalog #PE-121-1003) is required for clustering libraries on the cBot and paired-end sequencing on the HiSeq 2000, GAIIx, and in High Output mode on the HiSeq 2500. This kit is good for one run and contains the required primers for dual indexed sequencing (HP10, HP11, HP12).
The dual index primers (HP10, HP11, HP12) are included in MiSeq reagent kits and HiSeq 2500 Rapid reagent kits and do not need to be purchased separately for these types of runs.
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.
Running in triplicates is highly recommended to have high confidence in the results.
No reference genome is necessary for the MiSeq metagenomics workflow.
No. Cluster generation is performed on the MiSeq instrument as part of the run. No other instruments are required for sequencing on the MiSeq.
Final libraries are single stranded and cannot be quantified. Follow the normalization and pooling protocol before loading onto the MiSeq FGx.
The project scientist that is assigned to you works with you to submit representative test samples. These samples are quantitated at Illumina using PicoGreen to confirm sample concentration. Samples are genotyped on an Illumina array to determine genotyping quality. This data set is not for distribution. The data set is used exclusively for sample quality determination. After successful quality determination, the Illumina project scientist works with you to submit the production samples.
Yes. Illumina FastTrack Sequencing Services offer a human phasing sequence analysis service. See the Phasing Analysis Service FAQ for more details.
Yes. Illumina FastTrack Services offer a cancer analysis service for tumor-normal data set comparison. The standard offering is for a 40x normal and 80x tumor pair. See the Sequencing Service Process for more information.
The sequencing team performs downstream analysis for the cancer analysis service. For tertiary data analysis of standard whole-genome sequencing services, the Illumina Genome Network works with software partners who are experts in the field.
All Illumina sequencers use the Illumina patented reversible-terminator SBS chemistry. The NextSeq system employs the latest evolution in SBS technology: a novel 2-channel SBS method that supports reduced cycle time and data processing time.
No. Dual-indexed libraries sequence eight bases of actual index sequence rather than six bases plus one more for phasing calculations. For short sequences there are no phasing/prephasing corrections, so it is not necessary to sequence one additional cycle.
TruSeq Stranded Total RNA Sample Prep kits remove ribosomal RNA only. The Human/Mouse/Rat kits remove cytoplasmic ribosomal RNA and the Ribo-Zero Gold kits remove cytoplasmic plus mitochondrial ribosomal RNA.
Illumina supports all components of the workflow – library prep, sequencing, and software. Illumina bioinformatics support is well-trained on the Conexio software. In the event that Illumina support is unable to fully resolve a software question, we have escalation paths to internal Illumina experts and to experts at Conexio. If escalation is required, Illumina Technical Support will contact you directly with a resolution.
No testing has been performed on the effects of local proxies on BaseSpace access.
TruSight HLA uses long-range PCR for isolation and amplification of the HLA genes. These long-range amplicons range in size from 2.6 kb to 10.3 kb. Use a DNA sample that consists of at least 50% of the DNA greater than the size of the largest amplicon. In other words, if you are only interested in Class I genes (A, B, and C), then at least 50% of the DNA sample has to be larger than 4.2 kb.
TruSight HLA Amplicon Sizes:
Yes, the primer sequences are excluded from the analysis and allele assignment.
An internet connection is not required to use Assign 2.0. However, Assign 2.0 provides links to NCBI Blast, IMGT Allele Database, and AlleleFrequencies.net. These features require an internet connection.
Assign 2.0 is tested and supported on Windows. Virtualization software like Parallels or VMware allows you to run a Windows OS on a Mac or Linux. Therefore, it is possible to run Assign 2.0 through these virtualization technologies.
An internet connection is not required to use Assign. However, Conexio Assign allows you to launch NCBI Blast search on sequence reads directly from the interface. This feature requires an internet connection.
No. Assign is tested and supported on Windows. Virtualization software like Parallels or VMware lets you run a Windows application on a Mac or Linux. Therefore, it is possible to run Assign through Parallels on a Mac. However, this method is an unsupported function.
No. For germline variants, the software uses a simple rule set to predict the classification:
This pathogenicity autoscoring is only a suggestion. Review these predictions, the provided annotations, and all evidence for the variant to assign your final interpretation.
Yes, the software requires an internet connection. All software operations are conducted in the cloud.
Yes. The software supports VCF and gVCF file formats. For more information, see the BaseSpace Variant Interpreter Beta Online Help.
If you choose to use BaseSpace only for run monitoring and your samples are not indexed, a sample sheet is not required. If you want to use BaseSpace for data storage and analysis, a sample sheet is required. The sample sheet can be in either HiSeq Analysis Software format or CASAVA format. When using BaseSpace, combining indexed and non-indexed samples on a flow cell is not possible.
Yes. The Amplicon-DS workflow in the MiSeq Reporter software is used for the alignment and variant calling of TruSight Tumor data. The resulting VCF files can be imported into VariantStudio. Illumina recommends that you use the merged VCF file from the Amplicon-DS workflow. In these files, the variant call information from both the forward pool and reverse pool has been merged.
Yes. VariantStudio v2.1 and v2.2 support VCF and gVCF file formats. For more information, see the VariantStudio User Guide.
No. Illumina provides annotations from the respective annotation sources as is.
Illumina does not provide any recommendations as methods for ChIP pulldown and fragmentation are dependent upon individual antibodies and procedures. Please reference literature or other sources for recommendations.
Oligo Only Kits are not offered for the TruSeq DNA and RNA Library Prep kits.
Yes. Illumina offers a TruSeq Custom Enrichment kit. Reference the TruSeq Custom Enrichment Data Sheet for more information.
No, Illumina does not provide antibodies.
No, Illumina does not provide any classified variants. VariantStudio provides an empty Classification Database that you can populate by uploading classified variants from an external source or by manually classifying variants in samples that are being analyzed in VariantStudio.
No, Illumina does not provide classified variants. Using BaseSpace Variant Interpreter, you can upload classified variants from an external source or manually classify variants in samples that are being analyzed.
Illumina has not extensively tested any of the commercially available standards, and does not currently recommend any of them. In-house, Illumina uses standards generated in our own laboratory. Our methods for generating the standards are described in the following article:
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 different offsets. Background subtraction has a much smaller effect when you scan chips on the same scanner, and might not be necessary. Analyze a subset of data with and without subtraction, and choose the subset of data you prefer based on your results.
A library quality check on a Bioanalyzer is optional. See the appropriate library prep reference guide for library trace examples.
No. Filtering parameters vary depending on the application and objective of the investigator.
Use the following required pipettes and tips. Other pipettes and tips are not supported and can result in reagents not dispensing properly and run failure.
20 μl Volume:
200 μl Volume:
Yes. Illumina recommends the following of-the-shelf bisulfite conversion kits from Zymo Research.
D5001 50 reactions (single-column format)
D5002 200 reactions (single-column format)
D5004 2×96 reactions EZ-96 DNA methylation kit (deep-well format)
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.
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.
No. The two assays use different oligos.
In the random priming process to generate cDNA with the best coverage, this information is not retained. If originating strand information is required, please refer to the Directional mRNA-Seq Sample Preparation Guide or other published applications.
No, you perform variant classification. With VariantStudio, you can provide annotations that can be used to classify variants within VariantStudio. After you assign a classification category to a variant, the information is saved to the Classification Database so that it can be applied to the same variant observed in other samples. Classifications saved in the Classification Database can be applied automatically to other samples through the Apply Classifications from Database menu.
Yes, Illumina VariantStudio requires internet connection for annotating variants because the annotation database, known as the Illumina Annotation Service, resides in Amazon Web Services. After variants are annotated and saved in a project, an internet connection is not necessary.
No. VariantStudio is tested and supported on Windows 7 or later. Virtualization software like Parallels or VMware lets you run a Windows application on a Mac or Linux. Therefore, it is possible to run Illumina VariantStudio through Parallels on a Mac. However, this method is an unsupported function. It is recommended that you give your VM at least 2 GB of RAM and close any other programs.
cDNA libraries should be considered an equivalent to gDNA for clustering and sequencing steps, assuming an equivalent complexity of the sample.
Yes for a paired-end flow cell; no for a single-read flow cell. The dual-indexed workflow differs depending on flow cell type. For details on each workflow, see the NextSeq, MiSeq, and HiSeq Systems Indexed Sequencing Guide (part # 15057455).
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.
No, the HiSeq Analysis Software provides analysis of libraries prepared with the Nextera Rapid Capture exome enrichment kit and human Whole Genome Sequencing using only the hg19 genome as a reference. CASAVA provides analysis of additional applications such as RNA sequencing, Exome sequencing, targeted resequencing, and Whole Genome Sequencing using a more extended set of reference genomes available on Illumina.com's iGenomes page. (http://support.illumina.com/sequencing/sequencing_software/igenome.ilmn). More information on CASAVA is available here: http://www.illumina.com/software/genome_analyzer_software.ilmn#casava
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.
The data set generated by the NextSeq system is too large for on-instrument analysis. Data must be transferred to BaseSpace or a local server for secondary analysis.
The Ribo-Zero Globin kit supports human, mouse, and rat. Use of this kits with other organisms has not been tested and is not supported.
Please contact Illumina technical support with the rRNA sequence for your organism of choice to determine if your organism is compatible with the Ribo-Zero Globin kit. Please note that the analysis will be done in silico and does not guarantee rRNA removal.
The Ribo-Zero Plant kit will remove rRNA from a broad variety of plant RNA samples. It has been laboratory tested against Arabidopsis, maize, wheat, rice, corn, and soy bean. In silico testing, it has also demonstrated compatibility with Japanese honeysuckle (Lonicera japonica), mirror bush (Coprosma repens), moss (Physcomitrella), Tomato (Solanum lycopersicum), Vietnamese coriander (Persicaria odorata), and water clover (Marsilea vistita).
To check whether your organism of choice is compatible with this kit, see the Will a TruSeq Stranded Total RNA kit work with my organism? bulletin. If your organism of choice is not listed, use RNAMatchMaker to find which Ribo-Zero kit is compatible. Note that the analysis will be done in silico and does not guarantee rRNA removal.
The acquisition is done by a CCD camera through the top of the plate and through the optical adhesive sealer.
Use the Eco evaluation plate to test the performance of the instrument whenever needed.
The files uploaded as instrument health data are RunInfo.xml, RunParameters.xml, RTAComplete.txt, InterOp files, and RTAConfiguration.xml.
Dual-indexed runs on the HiSeq comprise 8 bp of index sequence rather than 6 bp plus a seventh for phasing calculations. For more information, see the user guide for your sequencing instrument.
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.
Perform this reaction in a thermal cycler to keep the temperature stable. Any PCR machine can be used for the bisulfite conversion reactions, but this reaction must be performed in the pre-PCR room.
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.
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/
All registered users receive an email notification when a new software is available. Details of changes between versions are provided in the release notes.
If a new version of the Illumina Annotation Service is released at the same time as a new software version release, the updates to the annotation database are specified in the software release notes. Otherwise, you receive an email notification informing you that a new database version is available. The email outlines the changes made relative to the previous annotation database version.
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.
Library concentrations are reported on the NeoPrep Control Software screen after the run is complete. In addition, the final report can be accessed using the Access Logs function on the Welcome Screen. The run report can also be saved to a network drive by specifying an alternate output folder location on the System Configuration screen. The information is also streamed to BaseSpace if run was set-up in BaseSpace Prep tab.
PCR cross-over events occur in the amplification of DRB1/3/4/5 when partially amplified products prime subsequent PCR reactions resulting in hybrid amplicons including template from more than one gene (eg, DRB1 and DRB3). On the assay side, we have reduced the number of PCR cycles to 30 cycles in an attempt to reduce the crossovers. However, bioinformatics has been employed to identify and remove any remaining crossovers. Forward and reverse reads are aligned independently to confirm that they match the same target. When bases from within a read can be matched to multiple targets in a sample, those bases are flagged and not used in the final consensus generation. The flagged bases can be observed in the coverage or read views and are marked as 0 quality.
TruSight HLA includes 4 boxes: 2 for pre-PCR(box 1 and 2) and 2 for post-PCR (box 3 and 4). Of each set, 1 box is shipped chilled and the other box is shipped frozen, as follows.
TruSight HLA v2 Sequencing Panel (24 Samples), catalog # 20000215, includes 3 boxes of reagents. Box 1 includes all the pre-PCR reagents including PCR mix, polymerase, buffers, and PCR primers. Box 1 is shipped on dry ice and is stored frozen at -25°C to -15°C. Box 2 contains purification and normalization beads used post-PCR and is refrigerated at 2°C to 8°C. Box 3 contains post-PCR buffers and tagmentation reagents. Box 3 is shipped on dry ice and is stored frozen at -25°C to -15°C.
TruSight HLA v2 Sequencing Panel (24 Samples Automated), catalog # 20005170, includes 4 boxes of reagents. Boxes 1 through 3 are identical to the ones previously described. The fourth box of auxiliary reagents includes additional purification beads required for dead volume minimums on many liquid handlers. The auxiliary reagents are refrigerated at 2°C to 8°C.
Base calling and quality scoring are performed by an updated implementation of Real-Time Analysis (RTA), called RTA v2, which includes important differences from RTA on other Illumina sequencing systems. For example, all processes are performed in memory to maximize processing speed, and configuration files and output file formats are different. For more information, see the NextSeq 500 System Guide or NextSeq 550 System Guide.
The workflow includes the following steps:
A beta value (β) is calculated for each CpG locus.
Each kit includes two flow cells and all required reagents, including clustering, SBS, paired-end, and indexing reagents. One kit provides consumables for a 150-cycle paired-end (2 x 150 bp) sequencing run plus indexing on both flow cell A and flow cell B.
With the release of the HiSeq X Reagent Kit v2, a new kit configuration called the 10-pack kit is available. The 10-pack kit includes flow cells and consumables for 10 dual flow cell runs or 20 single flow cell runs. The 10-pack is designed to support the preparation and sequencing of 4 flow cells at a time, and significantly reduces storage requirements.
The TruSight RNA Pan-Cancer Panel kit includes boxes 1-3 of TruSeq RNA Access. Box 4 is similar to the TruSeq RNA Access kit Box 4, but contains extra wash buffer (EEW) and PCR reagents needed for the single-plex protocol. Box 5 contains the oligo tube targeting the 1385 genes.
p = 10'(DiffScore*sgn(µcond-µref)/10)
For more information, see the Optimizing Coverage for Targeted Resequencing tech note.
Do the following to ensure that your network is performing fast enough for a particular run:
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.
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.
To create a report for all the analysis results in a project, or for a sample result, a report can be generated from the project page. A report for a sample result can also be generated from the sample details page. After a report is created, it can always be retrieved again from the project dashboard.
To create subpanels, generate gene lists in HGNC nomenclature for the regions of interest. Upload this subpanel gene list when importing VCF files into VariantStudio, and then filter variants based on the shortened gene list.
For reference, see the complete TruSight Cardio gene list
The sidebar in any of the Project screens contains all of the analyses with sample related to the selected project. Each analysis is individually selectable and immutable.
The manifest file is available for download from MyIllumina.
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.
Short fragments tend to create smaller clusters, allowing greater data density. The optimal fragment size for a single-read run is 150–300 bp. The optimal fragment size for a paired-end run is 250–500 bp.
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.
If these steps do not help reduce primer-dimer formation, redesigning the primers is necessary to obtain good results.
The TruSight Cardio *.bed file lists all targets of enrichment for this assay. Filter by gene name (HGNC nomenclature) or reference the coordinates of your loci of interest in the human genome reference (hg19 build). The gene list is also available on the product page.
Data can be visualized for run monitoring within MiSeq FGx Control Software. ForenSeq Universal Analysis Software can be used to monitor runs and visualize the results of analyses.
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.
To determine the chromosomal location for the potential/putative miRNAs, Illumina used the following procedure:
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.
BaseSpace Variant Interpreter is a web-based application. To make sure that you can access it through your company firewall, open port 443.
BaseSpace Interpreter and BaseSpace Hub use the same logon credentials for authentication, so logging on to 1 application logs you on to the other. After logging on to BaseSpace Interpreter, your account automatically links to your BaseSpace Hub account to allow import and viewing of variant call files stored there.
Data analysis is performed using the ForenSeq Universal Analysis Software.
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.
TruSeq Exome Enrichment data can be analyzed using the HiSeq Analysis Software enrichment workflow for HiSeq data or MiSeq Reporter Enrichment workflow for MiSeq data. For more information, see the HiSeq Analysis Software or MiSeq Reporter support pages.
If the run is being uploaded to Basespace or BaseSpace onsite, the samples can be analysed with either the BWA Enrichment or ISAAC Enrichment apps. Make sure that the correct manifest is selected before running the app.
Alternately, Illumina sequence base call output files (*.bcl) can be demultiplexed and converted to FASTQ format using the bcl2fastq converter software. The files can then be used for analysis with other third party software packages (eg, BWA and GATK).
Illumina suggests using a third-party methylation analysis solution such as Bismark, BSMap, or BS Seeker. See the Methylation Software section for more details.
Analyze HiSeq data using the HiSeq Analysis Software enrichment workflow for HiSeq data. Analyze MiSeq data using MiSeq Reporter. See the HiSeq Analysis Software or MiSeq Reporter support pages for more information.
Alternately, Illumina sequence base call output files (*.bcl) can be demultiplexed and converted to FASTQ format using the bcl2fastq converter software. The files can then be used for analysis with other third party software packages (eg, BWA and GATK). If the user is analyzing with the use of basespace, we recommend the use of BWA or Isaac enrichment analysis tools. If any subsampling is required, use the FASTQ Toolkit.
The GenomeStudio Methylation Module extracts beta values and provides clustering and normalization for Infinium MethylationEPIC array data. In addition, there are many freeware applications in Bioconductor, such as chAMP and RnBeads, which provide enhanced normalization and visualization options. Illumina does not support these freeware solutions directly.
The scan output default file format is GTC, which allows for direct analysis using the BlueFuse Multi software. If you prefer to use GenomeStudio for analysis, you can configure the instrument to generate IDAT files for export.
Assess the quality of genomic DNA by running an aliquot of the sample (approximately 10–100 ng) on a 1% agarose gel stained with SYBR Stain. High quality, intact genomic DNA appears as a high molecular weight band (> 10,000 bp) in the absence of a lower molecular weight smear. Low molecular weight smearing can indicate the presence of RNA or degraded DNA.
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.
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.
Use an Agilent Technologies 2100 Bioanalyzer to check the quality and intended size distribution of a Covaris sheared sample, the pre-enriched library, and the post-enriched library. For examples of bioanalyzer traces and library size distributions, see the TruSeq Exome Reference Guide.
Use an Agilent Technologies 2100 Bioanalyzer to check the quality and intended size distribution of a tagmented sample, the pre-enriched library, and the post-enriched library. For examples of bioanalyzer traces and library size distributions, see the library prep reference guide. Variation in the Bioanalyzer profiles is expected because it is dependent on the input DNA type.
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).
For more information, see the Nextera Low Plex Pooling Guidelines Technote.
Illumina uses a green laser to sequence G/T bases and a red laser to sequence A/C bases. At each cycle at least one of two nucleotides for each color channel need to be read to ensure proper registration. It is important to maintain color balance for each base of the index read being sequenced, otherwise index read sequencing could fail due to registration failure. For pooling strategies for a small number of samples, please refer to the "Adapter Tube Pooling Guidelines" section in sample prep guide for the kit you are using . Additionally it is recommended to create a sample sheet in the Illumina Experiment Manager (IEM) prior to performing sample prep in order to confirm appropriate index combinations not listed.
Choosing which primer mix to use depends on the application and legislation in a particular country. DNA Primer Mix A can be used for casework and database applications. Due to legislation in some countries, ancestral and phenotypic data cannot be stored in the database and DNA Primer Mix B can then only be used for casework.
The best way to confirm that an identified fusion is 'real' is to use an orthogonal approach. Additionally, assess the quality score and the chromosomal locations of the fusions to help indicate confidence.
In Configuration, select the LIMS tab and then select the Enable LIMS checkbox. Select the LIMS Server field and enter your LIMS server name using the onscreen keyboard. To generate a run data file (*.xml) for use with LIMS, select the Generate File with Experiment Information checkbox and specify a network location for the output. For more details, see the cBot System Configuration Guide (document # 1000000005301).
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 firstname.lastname@example.org, by phone at 1.800.809.4566 in the U.S., or on the Illumina website at http://www.illumina.com/support.
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.
There are numerous primer design tools commercially available for purchase or freely accessible via the web. These tools simplify assay design significantly.
Primers and probes for NuPCR, DBD, and HRM assays can be designed using Illumina's the web-based DesignStudio software (www.illumina.com/designstudio).
Other primer design tools are Primer Express (Applied Biosystems), Beacon Designer (Premier Biosoft) and Real Time Design(BioSearch Technologies). Numerous websites contain databases of validated primer sets, including RTPrimerDB (http://medgen.ugent.be/rtprimerdb/) and the Quantitative PCR Primer Database (http://web.ncifcrf.gov/rtp/gel/primerdb/)
If you design primers and probes manually, follow these criteria from the Primer Express manual:
Designating dual indices on the sample sheet depends on whether a MiSeq or CASAVA sample sheet will be used. For MiSeq sample sheets, each index is entered into its own column. For CASAVA sample sheets, the indices are input in the format of "Index1-Index2". Please see the MiSeq Sample Sheet Quick Reference Guide or CASAVA User Guide for more information.
Illumina recommends that you use the Illumina Experiment Manager (IEM) to generate your sample sheet. The appropriate index format is automatically entered based on the selected sample sheet type.
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.
The software provides a pass or fail indication for each positive and negative control sample. The pass/fail indication is based on the expected performance of the control.
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).
The used library card contains hazardous materials. Personal injury can occur through inhalation, ingestion, skin contact, and eye contact. Wear protective equipment, including eye protection, gloves, and a laboratory coat. Handle the used library card as chemical waste. Dispose of it in accordance with the governmental safety standards for your region. Refer to the safety data sheets (SDSs) for the consumables in the kit that you are using for detailed environmental, health, and safety information and dispose of according to your institution and other requirements. SDSs are available on the Illumina website at support.illumina.com/sds.html.
The positive sample tracking option is enabled by default. For more information about sample tracking and other configuration options, see the cBot System Configuration Guide (document # 1000000005301).
The Nextera XT DNA Sample Preparation Kit protocol is optimized for 1 ng of input DNA total. Illumina strongly recommends quantifying the starting genomic material. Nextera XT DNA Sample Preparation library preps use an enzymatic DNA fragmentation step and thus can be more sensitive to DNA input compared to mechanical fragmentation methods. The ultimate success of the assay strongly depends on using an accurately quantified amount of input DNA library. Therefore, the correct quantitation of the DNA library is essential.
To obtain an accurate quantification of the DNA library, it is recommended to quantify the starting DNA library using a fluorometric based method specific for duplex DNA such as the Qubit dsDNA BR Assay system. Illumina recommends using 2 μl of each DNA sample with 198 μl of the Qubit working solution for sample quantification. Methods that measure total nucleic acid content (e.g. nanodrop or other UV absorbance methods) should be avoided because common contaminants such as ssDNA, RNA and oligos are not substrates for the Nextera XT assay.
When using BaseSpace, sample sheet format can follow either HiSeq Analysis format or CASAVA format. For runs that require demultiplexing with either bcl2fastq 1.8.4 or CASAVA, a CASAVA-formatted sample sheet is required. This format is described in the bcl2fastq 1.8.4 User Guide (part # 15038058) and the CASAVA User Guide (part # 15011196).
Sample sheets for rapid runs include information for two lanes, as compared to eight lanes included in a sample sheet for a high output run. Sample sheets for rapid runs can be generated manually, using Excel or a text editor.
If you are using BaseSpace for data storage and analysis, a sample sheet is required for both rapid runs and high output runs. If using BaseSpace only for run monitoring and you are not indexing, a sample sheet is not required.
BaseSpace Variant Interpreter is a Software as a Service (SaaS) solution that is accessed through a web browser, so installation is not necessary. The minimum system standards are: Chrome version 49, Firefox version 45, and Internet Explorer 10, or later.
The BaseSpace Broker is designed to upload data to BaseSpace as soon as the data are generated on the HiSeq local drive. It will use as much bandwidth as is necessary to keep up with the data being produced. Under typical HiSeq run conditions, the upload of run data for storage and analysis will average less than 10Mbit/sec.
In most cases, throttling of the BaseSpace Broker data upload is not necessary. Throttling can be necessary if greater control over network bandwidth usage is required, such as sites where instruments share the network with other users or sites with limited upload speed. Throttling might be necessary in scenarios where the local network connectivity is temporarily lost and then restored. This interruption causes the BaseSpace Broker to suddenly consume more network bandwidth as it attempts to catch up with transfer of accumulated data. If no throttling is applied in such cases, the BaseSpace Broker might consume all available bandwidth on the network until the backlog of data are cleared. If throttling is applied and if the local network allows, Illumina recommends throttling to higher than the 10 Mbit/sec minimum specification. A recommended value of 20 Mbit/sec (approximately 3Mbytes/sec = 24Mbits/sec) allows the BaseSpace Broker enough bandwidth to recover, even if some delays in data transfer occur.
If throttling is needed, provide the following instructions to your local IT administrator:
Throttling of BaseSpace is performed on the HiSeq computer by application, rather than by IP address, as follows:
The run has met the Illumina specifications when the run metrics are within the thresholds and are colored green.
For information on how to name samples properly, see Create TruSight HLA Sample Plates and Sample Sheets with IEM (15069713 A).
We recommend using the TruSight HLA v2 Sample Sheet Template available from the TruSight HLA v2 Support Page. This MS Excel file generates sample names compatible with TruSight HLA Assign 2.0.
Using CASAVA: To merge data from different flow cells (different runs), use the configureBuild script in CASAVA v1.8.2. First, align the data (samples) from each flow cell separately using configureAlignment. Then, include each sample directory as an input directory in the configureBuild.pl command line. Input directories are specified by the -id option, as detailed on page 100 of the CASAVA v1.8.2 User Guide (Rev C).
If you are using CASAVA, note that Illumina is discontinuing distribution of CASAVA software to better support new products available on BaseSpace. BaseSpace features analysis options for a large array of NGS applications.
Using BaseSpace: BaseSpace includes a Sample Merge function that allows you to merge data from a single sample originating from different flow cells. This merging is performed before alignment analysis of the sample data.
Use the Decode File Client Utility to download Decode files directly from Illumina servers using standard HTTP protocol. You can download the Decode File Client and install it on a computer with access to the network location for the Decode file (DMAP) folder.
These files are preloaded in the software, and the location of the files is specified on the BeadChip Scan Configuration screen. From the NCS Home screen, select Manage Instrument, System Configuration, and then BeadChip Scan Configuration. Manifest files use the *.bpm file format, and cluster files use the *.egt file format.
For more information, see the NextSeq 550 System Guide.
The number of PCR cycles should be minimized to avoid skewing the representation of the library. Illumina recommends 15 cycles of PCR, which has been shown to provide the best performance in terms of coverage, reproducibility, and quantity of material. Although not recommended by Illumina, if you want to eliminate PCR, begin with a large amount of starting material (~5 μg) to generate equivalent yield as the standard protocol. Note that at least one cycle of PCR is required to open the forked adapters.
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.
The TruSeq DNA PCR-Free protocol does not contain a PCR enrichment step to select for completed ligation products, therefore quantitation must be done by qPCR as other methods will quantify fragments that may not have adapters at both ends and will thus not generate clusters or sequence. See the Consumables and Equipment section of the TruSeq DNA PCR-Free Library Prep Guide for the required KAPA Library Quantification Kit. Follow qPCR instructions included in the KAPA Library Quantification Kits for Illumina sequencing platforms Technical Data Sheet using the KAPA standard, with the modifications specified in the Validate Library sections of the TruSeq DNA PCR-Free Library Prep Guide.
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.
RTA 1.9 (on GA) or 1.10 (on HiSeq) or higher processes the controls. To visualize them, SAV (Sequencing Analysis Viewer) 1.7 or higher must be used. RTA produces a new InterOp metrics file called ControlMetrics; this is the file SAV uses to get control counts.
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.
Run the RNA-Seq workflow (FASTQ only) on the MiSeq and stream the data to BaseSpace. The BaseSpace RNA-Seq Alignment App analyzes data from the TruSight RNA Pan-Cancer Panel, providing a simple results summary that includes a fusion table, variant table, and gene expression table.
You can also use your own pipeline for analysis.
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.
Run data can only be uploaded to BaseSpace if the BaseSpace option is selected during run setup in the HiSeq Control Software. See the HiSeq 2500 System User Guide (part # 15035786) for information on setting up a run with a connection to BaseSpace.
For more information on BaseSpace, or to set up a free BaseSpace account, see https://basespace.illumina.com/home/index.
For sequencing of TruSight Cardio libraries on the MiSeq, use the MiSeq Reporter Enrichment workflow or the Generate FASTQ workflow. Illumina recommends preparing the sample sheet in the Illumina Experiment Manager (IEM). Libraries prepared with TruSight Cardio Sequencing kit (MiSeq, 12 samples) are dual-indexed, but only require a single i7 Index Read for demultiplexing. Set up the run for paired-end 151-cycle reads and a single 8 bp Index Read. FASTQ files can be uploaded to BaseSpace and analyzed using the BWA Enrichment App or the Isaac Enrichment App (v2.0 and v2.1 custom manifest workflow). Alternatively, use the MiSeq Reporter Enrichment workflow.
When using BaseSpace, set up the run in BaseSpace Prep tab. Select Nextera Rapid Capture from the dropdown menu under Library Prep kit to associate samples with the indexes provided in the TruSight Cardio Sequencing kit (NextSeq, 48 samples, Mid-output), E502, E503, E505, E506, and N701-12. Set up the run as a dual index paired-end 151-cycle sequencing run. FASTQ files streamed into BaseSpace can be analyzed using the BWA Enrichment App or the Issac Enrichment App (v2.0 and v2.1 custom manifest workflow).
When running the NextSeq in Standalone mode, enter the following parameters on the Run Setup Screen:
Read Type: Paired End;
Read Length: Read 1: 151; Read 2: 151; Index 1: 8; Index 2: 8
When the run is complete, use the bcl2fastq2 converter for demultiplexing and FASTQ conversion. Create a sample sheet for demultiplexing using Illumina Experiment Manager. Choose NextSeq > Nextseq FASTQ only > Nextera Rapid Capture Enrichment Sample Prep kit. Perform data analysis using third-party software.
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:
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:
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.
Use the Enrichment workflow to create a sample sheet formatted for analysis of TruSight One data. Refer to the IEM TruSight One or TruSight Rapid Capture Quick Reference Card for instructions on creating your sample sheet.
If you are interested in a project with Illumina Fast Track Microarray Services, contact your sales representative or get a quote through the Illumina website. An Illumina sales representative discusses your genotyping project and helps you select the product that best fit your needs. The sales representative also provides a quote. After you receive the quote, a project manager contacts you to discuss your microarray project and the sample submission process. For both test and production samples, the Illumina project manager ships you a thermal cooler that contains barcoded plates, sealing mats, and return labels. If necessary, a courier picks up your samples. There is no charge for shipping.
If you are interested in a project with Illumina FastTrack Sequencing Services, contact your sales representative or get a quote through the Illumina website. An Illumina sales representative discusses your sequencing project and helps you to select the product that best fits your needs. The sales representative also provides a quote. After you receive the quote, a project manager contacts you to discuss your sequencing project and the sample submission process.
Following the sequencing run, the NCS v2.0 software initiates an automatic post-run wash on the NextSeq 550 system, which takes about 90 minutes. When the wash is complete, the Home button becomes active and you can begin an array scan.
TruSeq Stranded mRNA NeoPrep libraries can be stored at -25°C to -15°C for up to 2 months.
In BaseSpace, click your Projects folder. Select Analysis and select the app session name that you saved your analysis under.
Use the DesignStudio table Export function to save designed regions, targets, and probes in *.bed file format. Exported *.bed files can be imported into a genome browser, such as the Broad Institute Integrative Genome Viewer (IGV), which can be downloaded and run on your local PC computer, or the online UCSC Genome Browser.
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.
Assign 2.0 breaks the sequence for each allele into 3 parts. The Core includes exons 2, 3, and 4 in Class I and exons 2 and 3 in Class II as well as sequence from known expression variants. The second part, Exons, includes all the other exons. By default, users only see sequence and phase mismatches from these 2 parts. It is simple to expand the view to see the third part, N-C, which includes the noncoding UTR and intron sequences. Even though the noncoding sequence is hidden from view by default, heterozygote positions within these regions are used for phase alignment. We also allow users to select the number of fields to review and report (Two, Three, or All). Ultimately, Illumina is supporting both IMGT and the 17th Workshop to solve the root of the issue, which is the missing noncoding reference.
Because run output has zipped BCL files, you must use the bcl2fastq v1.8.4 conversion software to perform BCL to FASTQ conversion on your local Linux analysis system. This tool is run on Linux and has the same syntax, options, and functions (including demultiplexing) as the configureBclToFastq.pl script of CASAVA. The only difference is that it can be used to analyze either zipped or non-zipped BCL files.
If you send your data to BaseSpace, BCL to FASTQ conversion and demultiplexing are performed automatically following the completion of the data upload.
When both alleles of a heterozygous position are different from the reference, as in a tri-allelic position, the variants are split into 2 lines and both variants are annotated.
For TruSeq Custom Amplicon, optimal probes are chosen using an algorithm that considers melting temperature (Tm), % GC, length, secondary structure, uniqueness in the genome, and the presence of underlying SNPs (based on dbSNP). For additional information, refer to the online help functionality in DesignStudio.
The ForenSeq DNA Signature Prep Kit contains two primer mixes to target specific regions of the genome. The mixes enable analysis of autosomal Y- and X-chromosome STR targets, identity-informative SNPs, with the option to include ancestry-informative, and phenotypic-informative SNPs. Libraries are prepared from an individual DNA sample, which can be multiplexed with libraries of other individual DNA libraries for sequencing on the MiSeq FGx Instrument.
Customer samples go through multiple quality control steps throughout the Illumina FastTrack Microarray Services workflow. The microarray team compares quality results to internal controls for accuracy verification. The microarray team takes these steps to ensure quality:
The Isaac aligner aligns reads by first identifying a small but complete set of relevant candidate mapping positions. The Isaac aligner begins with a seed-based search, using 32-mers as seeds. The initial single-seed search is followed by a multi-seed only for the reads that couldn't be placed unambiguously with a single seed. Speed up is achieved by sorting the reference index by the 32-mers. Improvement to accuracy is achieved by flagging of all the ambiguous reference positions in the index.
Following the seed-based search, selection of the best mapping among all the candidates is performed. For paired-end data sets, all mappings where only one end is aligned (orphan mappings) trigger a local search to find additional mapping candidates (shadow mappings) in the neighborhood defined by the expected minimum and maximum insert size. After optional trimming of low quality 3' ends and adaptor sequences, the possible mapping positions of each fragments are compared, taking into account pair-end information when available, possible gaps (using a banded Smith-Waterman gap aligner) and possible shadows. The selection is based on the Smith-Waterman score (using BWA, ELAND or user-defined scores) and on the log-probability of each mapping. The main speed-up comes from a parallel implementation of the gap aligner (using the SSE2 instruction set) and a shadow aligner optimized for short inserts. Further improvements could be achieved with AVX. The gapped alignment could be delegated to a coprocessor (e.g. Xeon Phi or GPU), however it is unclear if the benefit of large-scale parallelization would outweigh the cost of transferring the data between host and coprocessor.
Following alignment the fragments are sorted. Major speed-up in the sorting speed-up comes from efficient binning of the selected mappings, which greatly simplifies the sorting. Further analysis is performed to identify duplicates and optionally to re-align indels.
Illumina has designed the workflow to minimize the risk of contamination. However, Assign 2.0 flags any base call that is above the noise but below the minor allele frequency. These flagged positions require visual inspection and it is obvious during this process when contamination has occurred. The sequence reads from the contaminant can be blasted against the NCBI database by right clicking the read, making it easy to investigate the source of the contamination.
TruSight HLA v2 kits include 3 boxes and all pre-PCR reagents are included in a physically separate box (Box 1) from the post-PCR reagents (Boxes 2 and 3).
Bead-based normalization is a technique proprietary to Illumina and is an efficient method for normalization. This method eliminates the need for quantification and any associated equipment that make that process less onerous (eg, plate readers and robots). Bead-based normalization standardizes sample-to-sample and locus-to-locus variability. The bead-based normalization approach allows every amplicon to be normalized individually before pooling. Alternates to this approach involve quantitating every amplicon and normalizing individually based on the quantification. This process is so onerous that most recommend selecting a few amplicons to quantify and then normalizing based on the average. This method is risky as amplification varies from sample to sample and from allele to allele.
The TruSight HLA v2 protocol does not require a purification procedure. However, a bead-based purification method can be used. This method uses SPB (Sample Purification Beads) and RSB (Resuspension Buffer) from the TruSight HLA v2 kit. The TruSight HLA v2 Sequencing Panel (24 Samples Automated) includes an additional tube of SPB that can be used for this purification procedure.
1. Bring SPB to room temperature (~30 minutes).
2. Prepare fresh 80% EtOH.
1. Add 50 µl of input sample at 16 ng/µl of high-quality DNA to a 96-well midi plate.
2. Add 50 µl SPB to each sample.
3. Seal and shake at 1800 rpm for 2 minutes.
4. Incubate at room temperature for 2 minutes.
1. Place on a magnetic stand and wait until beads are fully pelleted and supernatant is clear (~2 minutes). Do not remove from the magnetic stand until instructed to do so.
2. With pipettes set to 200 µl, remove and discard 100 µl supernatant.
3. Add 200 µl fresh 80% EtOH to each well containing sample.
4. Incubate on magnetic stand for 30 seconds.
5. Remove and discard all supernatant from each well.
6. Repeat EtOH wash.
7. With a 20 µl pipette, remove all residual EtOH from each well.
8. Air dry on the magnetic stand for 2 minutes.
1. Add 52.5 µl RSB to each well containing sample.
2. Remove from the magnetic stand.
3. Seal and shake at 1800 rpm for 2 minutes.
4. Incubate at room temperature for 2 minutes.
5. Place on the magnetic stand until liquid is clear (~2 minutes).
6. Transfer 50 µl supernatant to a new plate.
Quantify using a fluorometric method, such as the Qubit BR assay. Then normalize each sample to 10ng/μl with at least 40 μl RSB per sample.
TruSight HLA v2 uses a dual indexing protocol using Illumina Nextera XT indexes. Each index is 8 bases in length and the Nextera XT index kits provide i5 and i7 indexes. Each fragmented template has an i5 and an i7 index incorporated. During data analysis, reads with perfect (16 of 16) and one mismatch (15 of 16) are assigned to a sample FASTQ. Libraries with 2 or more mismatches are not assigned to a sample and are not used in the final analysis since these reads are not included in the sample FASTQ file.
When both alleles of a heterozygous position are different from the reference, as in a tri-allelic position, the variants are split into two lines and both variants are annotated.
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.
The license key provided when ordering the kit expires 3 months from the order date. The license can be used for an unlimited number of samples, unlimited number of systems, and unlimited number of users. After the key has expired, previously saved projects (*.cgp) can be analyzed without an active license but new FASTQ files cannot be imported. To request a new key, contact Illumina Customer Service (email@example.com).
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.
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.
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.
Customer samples go through multiple quality control steps throughout the Illumina FastTrack Sequencing Services workflow. The sequencing team compares quality results to internal controls for accuracy verification. The sequencing team performs quality checks at each of these steps:
Detailed results are provided in our white paper located here: http://res.illumina.com/documents/products/whitepapers/whitepaper_iassc_workflow.pdf
In summary: Isaac currently has slightly lower sensitivity and specificity, though it is much faster than BWA/GATK. Efforts to improve Isaac are ongoing and new versions will become available as methods progress.
Total number of SNP conflicts, SNP conflict rate, and sensitivity (% of non N reference sites called) of Isaac and BWA+GATK
TruSeq Stranded Total RNA Sample Prep Ribo-Zero kits remove ribosomal RNA (rRNA) using a hybridization/bead capture procedure that selectively binds rRNA species using biotinylated capture probes. The probe:rRNA hybrid is then captured by magnetic beads and removed using a magnet, leaving the desired rRNA-depleted RNA in solution.
NextSeq 550 array data quality is comparable to the iScan system. For instance, both the iScan system and NextSeq 550 system can achieve > 99% SNP calls with a > 99% concordance (R^2) for the Infinium CytoSNP-850k BeadChip.
One pair of oligos is designed for each amplicon. Hybridization of these oligos to unfragmented genomic DNA occurs in a 96‐well plate, followed by extension and ligation to form DNA templates consisting of the regions of interest flanked by universal primer sequences. Using indexed primers supplied with the kit, DNA templates are then PCR amplified, pooled into a single tube, and sequenced on the MiSeq System. For additional details on the assay, please refer to the User Guide.
The NextSeq 500 system and NextSeq 550 system offer the same high sequencing data quality. There is no change to output or quality specifications between runs on the different systems.
This kit is similar to TruSeq Stranded Total RNA Library Prep because it enables RNA sequencing from low quality and FFPE-derived samples, and maintains strand information. Unlike TruSeq Stranded Total RNA Library Prep, this kit only captures the coding regions of the transcriptome, allowing much higher throughput and requiring lower sequencing depth.
This kit is similar to TruSeq Stranded mRNA Library Prep because it offers sequencing of the coding RNA and does not require ribosomal depletion.
This kit is compatible with low quality and FFPE-derived samples because it does not rely on the presence of a poly-A tail.
Because it enriches the coding transcriptome, the ribosomal RNA is washed away during the wash procedures.
Illumina does not have a specific schedule for updating the annotation database. This process largely depends on the cadence of updates from the different sources aggregated to create the annotation database.
Target Region Coverage is calculated as the proportion of bases in the requested Target Region that the designed amplicon sequences. The region that the designed amplicon sequences includes the nonvariable flanking probes and sequence in between.
Contamination appears as additional alleles in the results. Assign 2.0 calls the 2 most frequent alleles in the sample. All other base calls are either flagged or considered noise if they fall within the acceptable range. Base calls are flagged because they are higher than the noise, but have not achieved the frequency of the second most frequent base in the locus. Review flagged bases to quickly check whether contamination is present and to measure the degree present.
Furthermore, reads from these bases are available in the viewer. Right-click to blast these reads against the NCBI database to help determine the likely source of contamination.
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).
Multiple samples can be pooled together because of the use of unique index (barcode) sequences added during library preparation. Indexes allow the software to separate out the data for each individual sample after sequencing.
NextSeq 500 customers can purchase a package to upgrade to a NextSeq 550 system to enable scanning. The package includes a hardware change and a software update.
Array scanning is enabled out of the box on the NextSeq 550 system.
Where CE-based assays give the length of the STRs, Illumina technology provides, not only the length of the STRs, but any variation in the STRs and genotypes at the various SNPs. Illumina technology also allows multiple samples to be pooled together and sequenced in one run, instead of running samples one at a time.
STR and SNP data are shown in multiple formats in the ForenSeq Universal Analysis Software. On the sample details screen, the genotype and/or warnings for each locus is displayed A detailed presentation is available for each locus by selecting it. For STR loci, a table of alleles is provided as well as a bar chart of the alleles and their relative intensities (# of reads). For SNP loci, a table of alleles is provided as well as a pie chart showing the distribution of the alleles.
The TruSeq ChIP Sample Prep kit generates Paired End (PE), indexed libraries that are compatible with all Illumina sequencing platforms and can be multiplexed. The legacy ChIP-Seq Sample Prep kit generates Single Read (SR) and non-indexed libraries that are compatible with Genome Analyzer, HiSeq and HiScanSQ, but not MiSeq.
The beta value (β) uses the ratio of intensities between methylated and unmethylated alleles to estimate the methylation level of the CpG locus.
The first time the HCS 2.2 is launched, you will see a notification regarding instrument health data. This notification appears 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. However, instrument health agreement and notification is always available from Menu | Options | Tools, where you can also get more information and turn the option on or off.
Each template is flanked by index primers 8 bases in length, for a total of 16 bases. Only 15 of 16 and 16 of 16 matches are aligned to a sample and used in the analysis. All remaining specificity is managed bioinformatically. Off-target amplification occurs in the presence of certain other HLA genes (eg, HLA-Y and -H) and pseudogenes (eg, HLA-DRB6) and these sequences are identified and removed from the analysis. In addition, off-target amplification between HLA genes (eg, HLA-B primer amplification of HLA-C) can occur in the presence of certain alleles and this data alone cannot be used for reliable typing. Therefore, Assign 2.0 ensures that by assessing primer contribution, HLA-C allele assignment must be from the HLA-C primer amplifications.
The study number assigned to your samples is the next study number available in sequence.
The TruSight RNA Pan-Cancer protocol uses single-plex enrichment, whereas TruSeq RNA Access uses 4-plex enrichment. TruSight RNA Pan-Cancer uses single-plex enrichment to ensure even representation for all samples.
Additionally, TruSight RNA Pan-Cancer uses 14 PCR cycles during the enrichment step, whereas TruSeq RNA Access uses 10 cycles.
On average, ~2.75 Gb with JPEG files, which is the default setting.
You can start an array scan when the NextSeq 550 system completes the automatic post-run wash following a sequencing run.
You can start a sequencing run when the NextSeq 550 system completes the array scan.
The length of the upstream and downstream probe design can vary between 22 bp and 30 bp, regardless of the size of the amplicons being designed.
Final pooled libraries can be stored at -25°C to -15°C for up to 3 months.
Prepare HiSeq v4 SBS reagents the night before or on the same day of use only. Do not store reagents longer than overnight. For use on the same day, store prepared reagents on ice in the original bottle with the cap tightened. For use the next day, store prepared reagents at 2°C to 8°C overnight.
Libraries can remain at room temperature on a library card for up to 3 days after a run is complete.
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.
After amplification, linearization, blocking, and primer hybridization, you can store the flow cell in storage buffer at 2° to 8°C for up to 10 days. HiSeq X flow cells can be stored up to 48 hours.
For rapid flow cells, perform the sequencing run on the same day as sample loading.
Maintenance wash solution can be stored for up to 30 days at room temperature. In that 30-day period, the solution can be used up to three times. For the first use, assign each bottle and tube to a reagent rack position. Maintain those positions for the second and third uses to prevent cross-contamination.
Prepare rapid SBS reagents the night before or on the same day of use only. Do not store reagents longer than overnight. For use on the same day, store prepared reagents on ice in the original bottle with the cap tightened. For use the next day, store prepared reagents at 2°C to 8°C overnight.
The post-run wash takes 30 minutes and a maintenance wash takes about 1 hour.
The LED arrays are designed to outlast the life of the instrument and are covered by the instrument warranty.
Actual wash time for a maintenance wash is approximately 1.5 hours, not including flow check time.
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.
A 150-cycle run takes approximately 20 hours.
A 600-cycle run takes approximately 55 hours.
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.
Run duration depends on the number of cycles performed and the type of flow cell used. Run duration is the same for the NextSeq 500 system and the NextSeq 550 system. For more information, see the NextSeq System specifications page.
A water wash and maintenance wash each take approximately 1 hour. The maintenance wash protocol has changed to consist of only one step, washing the system with Tween 20 and ProClin 300, so there is no need to return to the instrument to reload wash solution or water.
Scanning takes approximately 40 minutes per BeadChip. The CytoSNP-850K scans at a rate of approximately 5 minutes per sample. The HumanCyto-SNP12 and HumanKaryomap-12 scan at a rate of 3.5 minutes per sample.
On the Cluster Station, it takes about 5 hours. On the cBot, clustering duration depends on the flow cell that you are using. HiSeq rapid flow cells take about 1 hour to cluster, and other HiSeq flow cells take about 3 hours. TruSeq v3 and GAIIx v2 flow cells are clustered in about 5 hours.
It takes 1.5-2 days (approximately 30 hours) from genomic DNA input until libraries are ready to load on the flow cell, including approximately 5 hours of hands on time. Nextera Rapid Capture Enrichment libraries can be loaded on the flow cell the afternoon of day 2 of the protocol.
It takes 2½ days for 8-24 samples from total RNA input until libraries are ready to load on the flow cell. This protocol includes approximately 11 hours of hands-on time.
It takes 9 hours from genomic DNA input until libraries are ready to load on the flow cell, including ~3 hours of hands on time.
The duration of a sequencing run depends on the type of run and number of cycles performed. See the MiniSeq specifications page for more information.
The post-run wash takes approximately 30 minutes.
The NextSeq software performs an automatic post-run wash after each successful sequencing run. The automatic post-run wash takes about 90 minutes.
NCS v1.4 introduces 2 manual washes: the Quick Wash and the Manual Post-Run Wash.
When you initiate a manual wash with NCS v1.3, the software checks that an automatic post-run wash was performed after the last run.
Preparing libraries takes approximately 9 hours. Sequencing and Real-Time Analysis (RTA) software tasks on the MiSeq FGx such as image processing, assigning base calls, and then designating a quality score for each base call takes approximately 30 hours. Aligning reads to provide sample information, along with optional steps such as generating reports, population statistics, sample comparison, phenotype estimations and bio-geographical ancestry estimations take approximately 1 hour.
Assign 2.0 does all the processing on the initial load of data. When the data are available for review, the power of the machine does not make a significant difference during review and even machines with minimum specifications perform quickly. The power of the computer has significance in the time it takes to perform the initial processing, which includes importing reads, aligning reads, phasing heterozygote positions, and assigning typing results.
Here are few different configurations and average processing time based on those configurations:
Number of Samples
Intel Core i7-5600 2.60 GHz
Intel Core i7-5600 2.60 GHz
Intel Core i7-5600 2.60 GHz
Intel Xeon X5560 2.80 GHz (x2)
Intel Xeon X5560 2.80 GHz (x2)
Intel Xeon X5560 2.80 GHz (x2)
~18-37 minutes per BeadChip
It takes about 45 minutes to change from a high output (HiSeq v4 or TruSeq v3) to TruSeq Rapid mode. It takes about 3 hours to change from Rapid Run to a high output mode. Time spent for mode switching is in addition to instrument washing performed at the end of each run.
The protocol runs for 7.5 hours, including library prep, quantification, and normalization. The run does not need intervention once started and libraries can remain on the instrument for up to 3 days before collection.
The protocol prepares libraries in 9.5 hours. The run does not need intervention after it is started and libraries can remain on the instrument for up to 3 days before collection.
Cluster generation is the first step in the sequencing run in which single DNA molecules are bound to the surface of the flow cell, and then amplified to form clusters. Cluster generation on the NextSeq system takes about 2 hours and 20 minutes.
The sequencing run on the MiSeq FGx takes approximately 30 hours to complete, including RTA primary analysis.
Both Index 1 (i7) and Index 2 (i5) are each 8 bp in length.
One BeadChip can be scanned at a time.
The TruSight One 9-sample kit provides sufficient reagents to prepare a maximum of three (3) enrichment reactions of up-to 3-pooled samples per enrichment. The TruSight One 36-sample kits provide sufficient reagents to prepare a maximum of three (3) enrichment reactions of up-to 12-pooled samples per reaction. Single sample enrichments are possible, but the reagent volumes are optimized for preparing three sets of pooled sample libraries at either the 3- or 12-plex levels mentioned previously.
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.
Currently, TruSeq Custom Amplicon supports up to 1536 amplicons in a single reaction. The number of amplicons in the multiplex reaction will vary based on the custom design and can range from 16–1536 amplicons. If more amplicons are desired (eg 3072), 2 reactions can be used with the same genomic DNA sample input. A single user can easily prepare upwards of ~150,000 sequencer-ready amplicons in a single day (96 samples x 1,536 amplicons).
Currently, TruSeq Custom Amplicon Low Input supports up to 1536 amplicons in a single reaction. The number of amplicons in the multiplex reaction will vary based on the custom design and can range from 16–1536 amplicons. A single user can easily prepare upwards of ~150,000 sequencer-ready amplicons in a single day (96 samples x 1,536 amplicons).
The kit contains three boxes.
Template generation, the process of identifying defining cluster positions over the entire flow cell surface, is performed during the first 5 cycles of the sequencing run. To detect a cluster during template generation, there must be at least 1 base other than G in the first 5 cycles.
MiSeq v3 kits are kitted for 150 cycles and 600 cycles.
HiSeq v4 SBS kits:
TruSeq v3 SBS kits:
HiSeq Rapid v2 SBS kits:
TruSeq Rapid SBS kits:
HiSeq v4 SBS kits:
TruSeq v3 SBS kits:
The NextSeq system can perform up to a 150-cycle paired-end run (2 x 150) using available NextSeq kits. Kits are available in sizes of 300 cycles, 150 cycles, and 75 cycles. Each kit includes additional cycles for index reads.
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.
Index reads for single-read libraries use 7 cycle reads. Illumina does not support 6 cycle index reads for single-indexed libraries.
Libraries are transferred onto the flow cell from a single reservoir on the NextSeq reagent cartridge, reservoir #10. Therefore, any libraries that can be pooled can be sequenced together on the flow cell.
The BaseSpace RNA-Alignment App with the STAR aligner calls a fusion if there are at least 3 unique reads that meet all the quality metrics. However, a high number of nonfusion supporting reads in that region would be expected to cause ‘noise’ that can affect fusion calling.
Mutational hotspots from over 48 genes associated with cancer are targeted with 212 amplicons.
This kit is available in a Set A and a Set B, each containing 12 indexes. When used together, sets A and B provide a total of 24 unique indexes.
There are a total of 24 unique indices: 12 in Set A and 12 in Set B.
LT kits: 12 unique single indices in Set A and 12 unique single indices in Set B
HT kit: 96 unique dual indices
The NextSeq flow cell contains 4 physical lanes. However, libraries are loaded onto the flow cell from a single reservoir. You can sequence a single library or multiple pooled libraries on the flow cell.
The MiSeq flow cell is a single-lane flow cell.
Up to 16 libraries can be prepared. If you run less than 16 libraries, it is recommended that the wells that do not have samples are the last ones in the row. RSB should be loaded into empty sample wells.
This kit is suitable for approximately 320 reactions when used for a TruSeq Low Input application.
This depends on the application. For expression profiling, 1–2M mapped reads is a generally accepted range. For discovery applications, an increase to 10–20M reads may be considered.
~3 million reads per sample is recommended. More or less reads may be required depending on the expression level of the fusion.
The TruSight HLA kit supports the preparation of 192 sequencing libraries. Each locus uses 1 library except for DRB1, 3, 4, and 5, which share a single library. If you are running all 11 loci per sample, prepare 8 libraries per sample and 24 samples per kit. If you are only interested in sequencing A, B, C, and DRB for each sample, create 4 libraries per sample for a total of 48 samples per kit.
The kit has enough reagents to process 24 samples. TruSight HLA v2 sequences and analyzes HLA-A, -B, -C, -DRB1/3/4/5, -DQB1, -DPB1, -DQA1, and -DPA1.
Because each well of the 96-well HT adapter plate is single-use only, only one sample per index pair can be generated.
When sequencing on the NextSeq system, you can pool 4 libraries containing 12 samples each (= 48 samples total). Make sure that each sample has a unique index combination.
1 to 96 samples can be processed at a time through the protocol. When processing fewer than the maximum number of samples per kit, store the aliquots of reagents for subsequent use instead of repeated thawing and freezing of the same reagent tubes.
8–96 single-source database or reference samples can be processed and pooled and 8–32 casework or evidentiary samples can be processed and pooled.
Each kit configuration allows for a certain maximum number of samples (24, 48, 72, 96) and all samples can be processed simultaneously. When processing fewer than the maximum number of samples per kit, store aliquots of reagents for subsequent use instead of repeated thawing and freezing of the same reagent tubes.
The MiniSeq flow cell is a single-lane flow cell, which requires that samples prepared for sequencing be combined in a single library pool. The library pool is loaded into a reservoir on the MiniSeq reagent cartridge before the run, and transferred onto the flow cell after the run is started.
The number of samples that can be combined in a library pool depends on the library prep method. See the documentation for your library prep kit.
This is dependent on the experiment plexity and MiSeq version. Illumina recommends using the TruSeq Targeted RNA Calculator to determine the number of samples that should be pooled together to achieve the depth of coverage required for your assay. Refer to the TruSeq Targeted RNA Expression tech note for information on how to design an experiment and determine the appropriate number of samples to be run on a flow cell.
TruSeq HT library prep kits support 96 samples. It is not recommended that the adapter plate undergo more than 4 freeze/thaw cycles. When less than the full set of 96 libraries are pooled and sequenced, it is important that libraries with compatible index combinations are used in the index pool. Please refer to the user guide for your TruSeq HT library prep kit for more information.
Each TruSeq ChIP Sample Prep kit contains enough reagents and adapters to process 48 samples. Set A and Set B each contain 12 unique indexes, with enough of each index sufficient for 8 individual samples.
The TruSeq DNA PCR-Free LT kits contain sufficient reagents for 24 samples and the TruSeq DNA PCR-Free HT kit contains reagents for 96 samples. Illumina recommends using the LT kit if processing less than 24 samples at a time and the HT kit if processing more than 24 samples. Both the LT kit and HT kit can be used with either the Low-Sample (LS) or High-Sample (HS) protocols.
The TruSeq Nano DNA LT Library Prep kits contain sufficient reagents for 24 samples and the TruSeq Nano DNA HT Library Prep Kit contains reagents for 96 samples. Illumina recommends using the LT kit if processing less than 24 samples at a time and the HT kit if processing more than 24 samples. Both the LT kit and HT kit can be used with either the Low-Sample (LS) or High-Sample (HS) protocols.
TruSeq DNA and RNA Sample Prep kits - Set A
TruSeq DNA and RNA Sample Prep kits - Set B
While Illumina supplies additional indexed adapters in the TruSeq Sample Prep v2 kits, the fill volumes are kept at 8 samples to minimize disruption to the protocols. The v2 kits are limited by other components to 48 samples per kit. This also offers more flexibility for how customers use the indexed adapters.
TruSeq Small RNA Library Prep kits are ordered as a core box and an index box. Each core box contains enough reagents to process 24 DNA samples and each index box contains 12 unique indexes, with sufficient index for two individual samples.
TruSeq Custom Amplicon and TruSeq Custom Amplicon Low Input have integrated sample barcodes that enable pooling of up to 96 samples per sequencing run. However, the actual number of samples that can be pooled together per sequencing run depends on the number of amplicons and the desired depth of sequencing coverage. An online calculator is provided in DesignStudio to help with these calculations.
Scanning and analysis of a 2-lane rapid run flow cell creates 2 swaths per surface on 2 surfaces per lane. Each swath is divided into 16 tiles. For a 2-lane flow cell, there are a total of 128 tiles per flow cell.
Scanning and analysis of a high output flow cell is performed in 3 swaths per surface on 2 surfaces per lane. Each swath is divided into 16 tiles. Therefore, an 8-lane flow cell contains 768 tiles per flow cell.
Scanning and analysis of a HiSeq 3000/4000 flow cell is performed in 2 swaths per surface on 2 surfaces per lane. Each swath is divided into 28 tiles. Therefore, each flow cell contains 896 tiles.
There are 2 types of flow cells available for the NextSeq system, the high-output flow cell and the mid-output flow cell. Both flow cells contain 4 lanes, but the lanes differ in width resulting in a different number of tiles.
TruSeq Custom Amplicon enables targeting of ~384 kb of cumulative DNA sequence (1536 amplicons x 250 bp each = ~384 kb).
TruSeq Custom Amplicon enables targeting of over ~650 kb of cumulative DNA sequence (1536 amplicons x 425 bp each = ~650 kb).
Going into the bisulfite conversion, at least 250 ng is required for the manual protocol, and at least 1000 ng is required for the automated protocol. Using less than the recommended amount does not guarantee R2 performance to meet specifications.
Refer to the DNA Input Recommendations section of the respective library prep reference guides.
Array scanning requires about 400 MB of free space on the NextSeq 550 hard drive. NCS v2.0 automatically manages disk space.
100 ng of input genomic DNA is required. Illumina highly recommends using a fluorometric-based quantification method for the input genomic DNA. For more information, see the DNA Input Recommendations section of the TruSeq Exome Library Prep Reference Guide.
Although the TruSeq Rapid Exome Enzyme is optimized to tolerate a level of DNA input variability, 50 ng of input genomic DNA is the target for optimum data. It is important to quantify the input genomic DNA accurately to generate a high-quality library of the correct size. Use a fluorometric-based quantification method for the input genomic DNA. For more information, see the DNA Input Recommendations section of the TruSeq Rapid Exome Reference Guide.
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.
|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|
See system specifications on the HiSeq 2500 Specifications page.
Use 10 ng of high-quality universal human reference total RNA as input. If starting with FFPE RNA, the sample input amount is based on sample quality. Use the percentage of RNA fragments > 200 nt fragment distribution value (DV200) as a reliable determinant of FFPE RNA quality.
Input Requirement Per Reaction
For more information, see the Evaluating RNA Quality from FFPE Samples tech note.
For successful library preparation, use an RNA isolation method that includes a reverse-crosslinking step and DNase1 treatment, such as the QIAGEN RNeasy FFPE kit or the QIAGEN AllPrep DNA/RNA FFPE kit.
For samples that border quality classifications, use the higher end of the input recommendation.
Use 10 ng of high-quality universal human reference total RNA as input. If starting with FFPE RNA, the sample input amount is based on sample quality. Use the percentage of RNA fragments > 200 nt fragment distribution value (DV200) as a reliable determinant of FFPE RNA quality.
Input Requirement Per Reaction
|Truseq RNA Access Library Prep: 20-40 ng|
|Truseq RNA Access Library Prep: 40-100 ng|
For more information, see the Evaluating RNA Quality from FFPE Samples tech note.
IMGT references are updated twice a year, in January and July, and are available from the TruSight HLA Support web page. Follow the instructions in the user guide to update the references in Assign. There are no automated updates.
IMGT reference updates are made available every 9 months, approximately 2 months after the IMGT release. They are available from the TruSight HLA v2 Support web page. Follow the instructions provided in the Assign 2.0 software guide to update the references in Assign 2.0. These updates are not performed automatically and can be installed at your convenience. Reference updates do not change the software and the software instance remains unchanged.
The NextSeq software performs an automatic post-run wash on the NextSeq 550 system after a successful sequencing run.
A maintenance wash is required every 10 days or when switching between high output and rapid modes. A water wash is required after each rapid run. After a high output run, you can choose between a water wash or a maintenance wash. Illumina recommends a maintenance wash.
We performed experiments that compared TruSight RNA Pan-Cancer to RNA Access, TruSeq mRNA, and TruSeq Total RNA. TruSight RNA Pan-Cancer was found to be concordant with the other applications (> 0.95 R2). High, medium, and low quality FFPE samples were also run and were found to be robust in performance (0.99 R2). In addition, comparisons between data from UHR (reference RNA) generated in different laboratories was highly concordant (R2 ≥ 0.97).
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.
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.
Illumina recommends that you check total RNA integrity following isolation using an Agilent Technologies 2100 Bioanalyzer with an RNA Integrity Number (RIN) value greater than or equal to eight.
Resuspension Buffer should be stored at -25ºC to -15ºC when first received. After the initial thaw, the reagent can be stored at 2ºC to 8ºC for use throughout the protocol.
The content for this panel was selected through collaboration with experts and key opinion leaders and by referencing publically available databases such as the Mitelman database and The Cancer Genome Atlas (TCGA).
The OncoArray consortium chose the selected SNPs specifically to develop a broad-use array platform that provides a detailed querying of:
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.
You can filter by Index in SAV (Sequencing Analysis Viewer) for indexed runs.
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.
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.
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.
Illumina has discontined the TruSeq DNA Sample Prep kits. The Sample Preparation: Kit Selector can assist in choosing whether the TruSeq DNA PCR-Free Library Prep or TruSeq Nano DNA Library Prep kit best fits your needs. In summary, the kit selected depends on available sample input amounts and the quality of data.
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.
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.
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.
See Chapter 5 of the GenomeStudio 2008.1 Framework User Guide, available on iCom and in the GenomeStudio Portal.
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.
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.
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.
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.
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 firstname.lastname@example.org for further investigation.
This amount of free space is required at the beginning of a run. The system assumes that data are transferred to the network copy of the run folder in real time. Therefore, 750 GB is the safe level to start a run. The software assumes that the run copies and deletes the files as they are processed, and that the connection to the network server can keep up with file transfer.
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).
Coverage of GC regions can be impacted by the model, settings, and performance of the thermal cycler used. Illumina has validated the Bio-Rad DNA Engine Tetrad 2, the Bio-Rad S1000, and the MJ Research PTC-225 DNA Engine Tetrad. Other thermal cyclers may differ in their performance across the genome.
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).
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.
TruSeq kits support many low plex pooling options across the entire plate. Some of these combinations are outlined in the Pooling Guidelines section of the appropriate TruSeq library prep guide. Customers can also design their own color-balanced pools, but we highly recommend that the Illumina Experiment Manager be used to check the color balance of user-designed pools
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 an upstream or downstream amplicon primer region overlaps an actual variant in the input genomic DNA, the assay can experience reduced specificity and/or uniformity relative to other amplicons in the multiplexed reaction.
No. VariantStudio v2.1 is programmed to use annotations from the previous version of the Illumina Annotation Service. It does not connect to the new version that is available with VariantStudio v2.2. If you want to use the new version of the Illumina Annotation Service, use VariantStudio v2.2 and reannotate any samples in projects from VariantStudio v2.1.
If I have custom content, can I get help with SNP selection? The project manager helps you design your SNP panel. In this collaborative process, the project manager uses Illumina design software to assess feasibility of SNP lists that you provide in the form of rsIDs or sequence submissions for novel SNPs or non-human panels. For each submission, the project manager provides a feasibility assessment as well as tag marker selection for haplotype blocks and gene annotations. After you select your final SNP list, the project manager takes the responsibility to order the beadpool for you.
You can import external sources of annotations into VariantStudio using the custom annotation feature. Check your HGMD Professional or HGMD public version license terms to make sure that such use is permitted.
No. If samples are reannotated in VariantStudio v2.2, all annotations for variants are replaced with the annotations from the newer version of the Illumina Annotation Service. If you do not want to lose previous annotations for these samples, create a project for the samples that you want to preserve with the old annotations.
No. You can start each flow cell independently from the other. Each flow cell can have a different number of reads and cycles.
Yes. The server storage size can store 1 run if there is network storage. Real-Time Analysis continues processing and resumes data transfer when the network is restored.
Yes. The server storage size can store 1 run if there is network storage. Real-Time Analysis continues processing and resumes data transfer when the network is restored.
No. VariantStudio v2.2 is programmed to use annotations from the version of the Illumina Annotation Service that was updated for VariantStudio v2.2.
You need 3 thermal cyclers with heated lids per 192 libraries, a microplate shaker up to 1800 rpm, and 96-well plate magnets for the bead normalization steps. A microplate heater is optional (thermal cyclers can be used instead).
Assign 2.0 is a new installation and can be run side-by-side with Assign 1.0 for TruSight HLA.
No. BaseSpace Variant Interpreter is a Software as a Service (SaaS) solution with a graphical user interface that allows variant exploration, annotation, filtering, and reporting without bioinformatics expertise.
Yes. HCS 2.2 is available for all customers.
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.
A dilute solution of NaOCl is required for the automatic post-run wash.The required NaOCl is included in the reagent cartridge provided in the NextSeq 500/550 Kit v2 and the TG NextSeq 500/550 Kit. However, if you are using the original NextSeq 500 Kit (v1), load 3 ml NaOCl in reservoir #28 before loading the reagent cartridge.
Requirements for NaOCl differ for manual instrument washes depending on the version of control software you are using:
The Directional mRNA-Seq Sample Preparation Guide provides instructions to use the Illumina Small RNA adapters, which are specific to the 3' and 5' fragment end, to generate a stranded library with the original Illumina mRNA-Seq kit. The protocol has not been optimized for the TruSeq RNA Sample Prep Kit and Illumina does not recommend this use of the TruSeq RNA Sample Prep Kit.
hg19 + refseqs transcript model
dbSNP build 137, common SNPs ≥ 1% MAF
mm9, mm10 + refseq transcript model
dbSNP build 128 for mm9, dbSNP build 137 (common SNPs) for mm10
dbSNP build 125 for rn4
Yes, TruSight HLA v2 is considered a shotgun sequencing approach in which the HLA targets of interest are randomly fragmented, sequenced, and the sequences of the fragments are assembled using bioinformatics.
No. Illumina VariantStudio was is a point-and-click software application that enables variant data exploration, annotation, and filtering without requiring bioinformatics expertise.
No, the reagent blank is made by the kit user.
When connected to BaseSpace, a sample sheet is not required. Library and indexing information is entered on the BaseSpace Prep tab before the run, and the information is passed to the NextSeq system. Available run names appear on the instrument screen during the run setup steps.
However, if the instrument is configured to run in standalone mode (not connected to BaseSpace), use Illumina Experiment Manager (IEM) v1.8.2, or later, to create a sample sheet.
For runs on the HiSeq, HiScanSQ, or GAIIx, creating and loading a sample sheet at the start of the run is optional. However, using a sample sheet allows you to view data shown on the indexing tab in the Sequencing Analysis Viewer (SAV) during the run. If you do not load a sample sheet at the start of a run in HCS, you will not be able to view indexing data in SAV. When analyzing indexed samples using CASAVA v1.8.2, a sample sheet is required. MiSeq runs require a sample sheet when setting up the run in MCS.
Illumina recommends that you create the sample sheet using the Illumina Experiment Manager (IEM) prior to performing library prep in order to confirm appropriate index combinations.
No, a wash is not required after a scan. However, if the system has been dry for 7 days, a wash is required before proceeding to another scan.
All library prep processes occur within the tightly controlled environment of the NeoPrep library card. The NeoPrep instrument is dry and performs no liquid handling. The library card is disposed of at the end of the run, therefore no washes are required. Routine maintenance procedures are included in the NeoPrep Library Prep System Guide.
Contamination is always a concern with PCR. Illumina recommends setting up PCR1 in a Pre-PCR facility. Illumina recommends using two areas in the post-PCR area: one for setting up PCR2 (no libraries present in this area) and a separate area for the library purification, bead-based normalization, and MiSeq FGx set-up.
Only LT adapters are methylated and the polymerases used in the TruSeq kits are not appropriate for bisulfite applications.
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.
No. TruSight HLA is configured for flexibility. The TruSight HLA kit supports the preparation of 192 sequencing libraries. Volume of certain locus primers has been increased to support sequencing more of these loci.
Libraries supported for each locus:
Examples: If you prepare all 8 libraries (11 loci) per sample, the TruSight HLA kit supports 24 samples. If you run only A, B, C, and DRB1/3/4/5, the TruSight HLA kit supports 48 samples.
We recommend running all the loci on each sample as the pooling post-amplification minimizes the time and cost savings of running fewer loci. If you must run fewer loci, adjust reaction volumes to compensate for the missing loci.
Currently this is not supported by the Eco system. Check back periodically and keep an eye on the monthly Illuminotes newsletter for updates.
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.
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.
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.
No. Always perform a wash when a wash is due. It is not possible to proceed to scanning or sequencing until a wash is performed. Instrument washes are required every 7 days, even when the instrument is used for array scans, since the instrument is in a dry state. Regular washes help maintain the instrument fluidics system.
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.
Yes, you can select 3 types of workflows on NeoPrep:
If you are not using the on board quantification, libraries can be quantified using a fluorometric quantification method that uses dsDNA binding dyes or qPCR. For more information, see the appropriate library prep reference guide.
Samples can be quantified prior to pooling, but this process is often inaccurate. At high plexity, it may be more effective to pool using equal small volumes prior to gel purification then purify, amplify and quantitate the pooled library. Once the pool is sequenced, any samples that did not give sufficient coverage can be re-pooled and sequenced.
Quantification is performed before pooling the libraries and must be done using qPCR. Other methods will quantify fragments that may not have adapters at both ends and will thus not generate clusters or sequence. Quantification by methods other than qPCR will be inaccurate. It is possible to quantify after pooling if all DNA samples are of similar quality, but this requires very consistent yields and should not be attempted by a new user. See the TruSeq DNA PCR-Free Library Prep Guide for details.
Yes, it is good practice to requant after the dilution is made to confirm the amount before starting the assay.
If you use at least 250-1000 ng DNA for the bisulfite conversion, requantification is not necessary. It is critical to quantify the input DNA concentration with PicoGreen to make sure that you add sufficient DNA to the bisulfite conversion reaction. Bisulfite conversion renders DNA less complementary, so much of the DNA are denatured and more difficult to quantitate accurately.
Assign has undergone standard software development and testing requirements applied to all Illumina software tools developed for Research Use Only.
The Assign software has not been submitted for regulatory approval. The software has not undergone analytical validation or clinical validation required for software tools that are regulated as a medical device. Therefore, it is important that you follow procedures for validation of the software according to your Institution, Local, State, and Federal guidelines.
BaseSpace Variant Interpreter has undergone the standard software development and testing requirements that are applied to all Illumina software tools developed for Research Use Only. Illumina has not submitted the software for regulatory approval and it is not a medical device. It is important that you follow procedures for validating the software per institution, local, state, and federal guidelines.
Currently, the Eco system is only available in a 48-well format.
One complete sample loading dock is provided with each Eco system.
To purchase additional docks:
2. Click the Options & Accessories tab
3. Scroll down to the Eco sample loading dock field
4. Place your order
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.
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.
Yes, the same controls types are present.
The sequencing team runs each of your samples in parallel on the HumanOmni2.5 genotyping BeadChip. You receive a file of the genotyped SNPs in VCF format. Illumina GenomeStudio software directly outputs the VCF file. The calls are obtained by applying the product standard cluster file to your data. For projects larger than 100 samples, Illumina recommends that you recluster on the project samples, check SNP performance, and re-export the genotype calls.
Illumina also provides intensity data files (IDATs) and a sample sheet. You can combine these files with the product definition files (beadpool manifest .bpm, and standard cluster file .egt) that are found on the Illumina support website to recreate a GenomeStudio project from the source data. This step allows you to visualize and assess SNP and sample performance.
Yes, the TruSeq DNA PCR-Free Library Prep kits will give better coverage for difficult to sequence regions. Please see the TruSeq DNA PCR-Free Library Prep Kit Datasheet and example datasets for more detailed information.
TruSight HLA Assign 2.0 software is included in the TruSight HLA v2 kits. Upon order of TruSight HLA v2, you receive an email with the link to download the Assign 2.0 software and a license file attached to the email. If there is no email associated with the order or the email goes to the wrong person, contact Illumina Customer Service (email@example.com) and provide the order number to receive a license.
Yes, BaseSpace can be used for the analysis of TruSight One runs. Illumina recommends using the Illumina Experiment Manager and select Targeted Sequencing and the subsequent Enrichment workflow. Download VCF files from BaseSpace projects to filter variants using the VariantStudio software.
No. The VariantStudio software was developed as a Research Use Only tool.
Illumina VariantStudio has undergone standard software development and testing requirements applied to all Illumina software tools developed for Research Use Only.
Illumina has not submitted the Illumina VariantStudio software for regulatory approval. The software has not undergone analytical validation or clinical validation required for software tools that are regulated as a medical device. Therefore, it is important that you follow procedures for validation of the software according to your Institution, Local, State, and Federal guidelines.
Illumina recommends performing the gel size selection step as it is designed to remove unligated adapters, as well as any adapters that might have ligated to one another, and selects a narrow 250–300 bp size-range of DNA fragments for ChIP library construction appropriate for cluster generation.
Yes. The incorporation reagent included in the HiSeq v4 SBS kit appears bluer, instead of the purple color of incorporation reagents included in the TruSeq v3 SBS kit.
No, indexing primers are specific to the SR or PE cluster kit because the indexing workflow is different for each flow cell type. For details on indexing workflows, see the NextSeq, MiSeq, and HiSeq Systems Indexed Sequencing Guide (part # 15057455).
No. The HiScanSQ runs HCS 1.5.15 and is not upgradeable.
If you are using CASAVA, it is compatible. However, bcl2fastq v1.8.4 must be used in place of the configureBcl2fastq step in CASAVA. The output of bcl2fastq v1.8.4 is in the fastq.gz file format organized into project and sample directories as specified in the sample sheet. This output is compatible with the configureAlignment and configureBuild components of CASAVA v1.8.2. The sample sheet format required for bcl2fastq v1.8.4 is equivalent to CASAVA v1.8.2 sample sheet format, and is described in the bcl2fastq v1.8.4 User Guide (part # 15038058).
If you are not using CASAVA, note that Illumina is discontinuing distribution of CASAVA software to better support new products available on BaseSpace. BaseSpace features analysis options for a large array of NGS applications.
LIMS support is not currently available for FFPE samples on the Infinium MethylationEPIC BeadChip.
LIMS support is available for the Infinium HumanMethyation450 BeadChip and Infinium MethylationEPIC BeadChip.
No. Illumina recommends purchasing a 600-cycle kit and discarding the excess reagents after the run. Because of the increased cluster density enabled by v3 runs, this is more economical than running fewer samples with MiSeq v2 reagents.
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.
The new index adapter design enables PCR-free protocols. (A single cycle of synthesis is required to separate the forked adapter.) However, for applications that require higher amounts of input for sequencing, Illumina recommends 10 cycles of PCR (with an optional titration for potential cycle reduction and to optimize throughput). The TruSeq DNA LT/HT kits (FC-121-2001, FC-121-2002, FC-121-2003) use 10 cycles of PCR. The TruSeq DNA PCR-Free LT/HT kits (FC-121-3001, FC-121-3002, FC-121-3003) are optimized for PCR-free applications. These kits and protocols are not interchangeable. Please see the TruSeq DNA PCR-Free HT Sample Prep Kit support pages and the TruSeq DNA PCR-Free LT Sample Prep Kit support pages for more information.
No, the pricing remains the same.
There is a 24 kb limit of contiguous sequence that can be entered for a given target region so as not to overwhelm the design server memory resources. For designs spanning large stretches of DNA, Illumina recommends TruSeq Custom Enrichment, a hybridization-based enrichment for targeted resequencing.
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.
Processing fewer than 8 samples at one time, including positive and negative controls, can cause problems with pipetting accuracy, due to the small volumes used when preparing the master mix.
An applicator tool (squeegee) is included with each Eco sample loading dock.
No, there is no on-instrument analysis solution.
Yes. The ‘Avoid SNPs’ design option will also avoid indels from the given variant database being used (eg dbSNP for human designs).
TruSight HLA uses long-range PCR for isolation and amplification of the HLA genes. These long-range amplicons range in size from 2.8 kb to 10.3 kb. Illumina recommends that the DNA sample consist of at least 50% of the DNA greater than 20 kb because overly fragmented DNA affects the long-range PCR.
TruSight HLA v2 Amplicon
Projects that are in-progress may keep their current configuration and workflow rather than changing protocols in the middle of the study. Additionally, pooling reads from paired-end stranded runs is equivalent to the same number of reads from non-stranded single read runs (i.e., 2 million reads each of read 1 and read 2 pooled together (4M total) is identical to 4 million reads of read 1 of a non-directional library).
MiSeq data sets are available on the BaseSpace Public Data page.
Datasets are available on the BaseSpace Public Data page.
TruSeq RNA Access, which forms the backbone of TruSight RNA Pan-Cancer, has been automated by other automation providers.
If you are interested in automating this workflow, contact your account manager.
Introducing too much DNA onto the flow cell results in a raw cluster density over the recommended 1.4M/mm2 threshold. Overclustered flow cells can result in poor quality data, as individual clusters cannot be adequately resolved and low numbers of clusters pass filters to be used in final output. It is recommended to target < 1.4M/mm2 to ensure high-quality sequencing performance.
Training is highly recommended but not required. TR-204-0024 is on-site customer training for TruSight HLA v2 library preparation. Customers receiving on-site library prep training have had a much better experience and have implemented the solution faster than labs not receiving training.
Sequencing 550 bp inserts is not currently compatible with the NextSeq 500.
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.
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.
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 firstname.lastname@example.org for pricing and assistance.
To confirm fusion calls, perform additional investigation or orthogonal testing. Fusion transcripts between nearby genes on the same chromosome and strand can be a result of read-through transcription rather than genomic translocation. To reduce these calls, the fusion software is optimized to filter read-through transcripts, but can result in filtering of biologically relevant fusions between adjacent genes (eg, STIL-TAL1). Also, fusions called between genes of high homology, such as a gene and its pseudogene, can be artifacts of multiple alignment instead of genomic rearrangements. Assessing the quality score, chromosomal location, and confirming with an orthogonal approach is recommended.
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.
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.
RNA libraries should routinely be run as single read (with an index read as desired). TruSeq libraries can be run as paired-end if desired (for example, for directional RNA custom preparations), but this is expected to offer no advantage and significant expense with small RNA libraries.
Illumina recommends locating the instrument in a post-PCR laboratory, because the final product collected from the instrument is amplified libraries.
RNA that has DNA contamination results in an underestimation of the amount of RNA used. Including a DNase step with the RNA isolation method is recommended to prevent DNA contamination in the final libraries.
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.
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 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.
Example Fluorophores Detected
SYBR Green I, FAM
Illumina recommends using Coriell Human-1 DNA (NA18507) or Promega Human Genomic DNA (G3041) as a positive control for this protocol.
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:
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.
A list of the Illumina barcodes and their corresponding HapMap IDs is available upon request from email@example.com.
This is the HumanHap550v1 product.
While there is enough coverage on the Nano flow cell for 12 samples for each to achieve 100x mean depth at each locus, optimal library preparation and sequencing conditions are required. If the library preparation or sequencing performance results in lower data output, you may not get sufficient sequencing coverage to achieve 100x mean depth of coverage for every locus.
Supported BeadChips include the CytoSNP-850K, HumanCytoSNP-12, and HumanKaryomap-12.
We recommend 10 ng of high quality genome DNA. For FFPE samples the input amount depends on ∆Cq. For more information, see the TruSeq FFPE DNA Library Prep QC Reference Guide (document # 1000000002137).
Illumina recommends 10ng of high quality genome DNA. For FFPE samples the input amount depends on ∆Cq. For more information, see the TruSeq FFPE DNA Library Prep QC Reference Guide (part # 15067391).
For Infinium arrays, Illumina recommends use of PicoGreen reagent for DNA quantification. UV-based methods may can overestimate DNA concentration by 2–10 fold.
TruSeq ChIP libraries are compatible with all Illumina sequencing platforms, including MiSeq, HiSeq, HiScanSQ and Genome Analyzer.
CASAVA and (MSR) can be used for demultiplexing. However, they are not intended for TruSeq ChIP analysis.
There are 3 MiSeq reagent kits that support ≥ 500 cycles (2 x 250 bp).
|Sequencing Kit||Catalog Number||TruSight HLA Samples||Run Time (Hours)|
|MiSeq Reagent Kit Nano v2 (500 cycles)||MS-103-1003||6||28|
|MiSeq Reagent Kit v2 (500 cycles)||MS-102-2003||24||39|
|MiSeq Reagent Kit v3 (600 cycles)||MS-102-3003||Untested||60|
Nextera Rapid Capture Exome Enrichment (8 rxn × 1 plex)
Nextera Rapid Capture Exome Enrichment (8 rxn × 3 plex)
Nextera Rapid Capture Exome Enrichment (8 rxn × 6 plex)
Nextera Rapid Capture Exome Enrichment (8 rxn × 9 plex)
Nextera Rapid Capture Exome Enrichment (2 rxn × 12 plex)
Nextera Rapid Capture Exome Enrichment (4 rxn × 12 plex)
Nextera Rapid Capture Exome Enrichment (8 rxn × 12 plex)
Nextera Rapid Capture Expanded Exome Enrichment (2 rxn × 12 plex)
Nextera Rapid Capture Expanded Exome Enrichment (4 rxn × 12 plex)
Nextera Rapid Capture Expanded Exome Enrichment (8 rxn × 12 plex)
Nextera Rapid Capture Custom Enrichment (48 Samples)
Nextera Rapid Capture Custom Enrichment (96 Samples)
Nextera Rapid Capture Custom Enrichment (288 Samples)
For STR typing, ForenSeq Universal Analysis Software presents a locus quality control indicator for the following conditions:
- More alleles are present than expected
- Alleles are not balanced
- An allele is below the interpretation threshold but above the analytical threshold
- The number of reads was not enough to make a call
- Stutter is present above the allowable level for the locus
For SNP typing, ForenSeq Universal Analysis Software presents a locus quality control indicator for the following conditions:
- Alleles are not balanced
- An allele is below the interpretation threshold, but above the analytical threshold
- The number of reads was not enough to make a call
# of Samples
TruSight Cardio Sequencing Kit for MiSeq and MiSeqDx
MiSeq v2 chemistry, MiSeq reagent kit included
TruSight Cardio Sequencing Kit for NextSeq
NextSeq v2 chemistry, mid output
TruSight One Sequencing Panel (9 samples). Includes MiSeq reagents.
TruSight One Sequencing Panel (36 samples). Sample Prep only.
TG TruSight One Sequencing Panel (9 samples). Includes MiSeq reagents.
TG TruSight One Sequencing Panel (36 samples). Sample prep only.
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.
The cBot 2 offers positive sample tracking for the cluster generation step of the sequencing workflow. The instrument records the barcode ID of the reagents, flow cell, library template, and any custom or additional primers used for a run. You can configure your cBot 2 to share those IDs with LIMS, or work in standalone mode.
The HiSeq 3000 is designed as an open platform intended to support a broad array of applications. To date, validated applications are whole genome sequencing, RNA sequencing, and exome sequencing. For more information, see HiSeq 3000/HiSeq 4000 Applications.
The HiSeq 4000 is designed as an open platform intended to support a broad array of applications. To date, validated applications are whole genome sequencing, RNA sequencing, and exome sequencing. For more information, see HiSeq 3000/HiSeq 4000 Applications.
The HiSeq X system is intended for large-scale whole-genome sequencing projects using either the TruSeq Nano DNA (HT or LT) or the TruSeq DNA PCR-Free (HT or LT) library prep kits. Other applications are not compatible or supported on the HiSeq X system.
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.
The MiniSeq System enables small genome, amplicon, targeted enrichment, and RNA sequencing of low library volumes with associated analysis options using BaseSpace Apps or Local Run Manager analysis modules. For more information, see the MiniSeq System applications page.
For a list of specific library prep kits associated with Local Run Manager analysis modules, see Compatible Libraries on the Local Run Manager support page.
The ForenSeq DNA Signature Prep Kit is used for two primary applications: Criminal Cases and Database, but can also be used in other applications, such as Missing Persons, Mass Fatality, and Bio-geographical Ancestry.
The MiSeq supports a large portfolio of sequencing applications. See the MiSeq Applications page for more information.
Illumina FastTrack Services combine high-performance Illumina platforms with expert Illumina scientists. Illumina FastTrack Services deliver high-quality genotyping and sequencing data to support your research projects. You reap the benefits of Illumina technology with a personalized service that delivers your data quickly and at a reasonable cost.
Illumina FastTrack Services offer a broad range of services, from whole-genome genotyping to custom content genotyping, human whole-genome sequencing, and human phasing analysis services.
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.
The table below includes Illumina’s minimum hardware recommendations to run GenomeStudio software.
|Parameter||Sequencing Data Analysis||Microarray Data Analysis||Microarray and Sequencing Data Analysis|
|CPU Speed||Intel Pentium 2.0 GHz or faster||Intel Celeron Duo or faster||Intel Celeron Duo or faster|
|Memory Size||4 GB or more||8 GB or more||8 GB or more|
|Hard Drive||250 GB or larger||100 GB or larger||250 GB or larger|
|Video Display||1,280 x 1,024||1,280 x 1,024||1,280 x 1,024|
|Operating System||Windows XP, Vista, or Windows 7||Windows XP, Vista, or Windows 7||Windows XP, Vista, or Windows 7
|Specific OS Requirements||Microsoft .NET Framework 3.5||Microsoft .NET Framework 3.5||Microsoft .NET Framework 3.5|
|Network Connection||1 GbE or faster||1 GbE or faster||1 GbE or faster|
Accurate library quantification is needed to achieve even pooling for enrichment as well as the recommended raw cluster densities. Inaccurate quantitation and pooling can result in higher representation of some samples compared to others in the same pool. Accurate quantification of the final library before loading is needed to reach the recommended raw cluster densities. Illumina recommends using the same diluted library for both quantitation as well as clustering. If cluster densities are lower than expected, Illumina recommends checking the library size used in the concentration calculation using a Bioanalyzer trace and adjust accordingly.
BaseSpace is the preferred analysis solution for the HiSeq 3000. For third-party analysis packages, you can use the bcl2fastq converter. CASAVA and HAS are not supported.
BaseSpace is the preferred analysis solution for the HiSeq 4000. For third-party analysis packages, you can use the bcl2fastq converter. CASAVA and HAS are not supported.
There are eight safe stopping points in the TruSeq RNA Access Library Prep protocol:
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).
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.
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).
See TruSeq Custom Amplicon Low Input Library Prep Reference Guide for more details about the new workflow.
There are a number of third party solutions available for the analysis of ChIP data. These include, but are not limited to, MACS, Avadis, and Partek. Please note that Illumina cannot provide support for the use of third party software; please contact the software resources directly with any questions regarding the analysis and use of their software. Additional literature references can be found on the Illumina Epigenetics webpage.
Illumina recommends 50 μl input DNA at 10 ng/μl.
For more information, see DNA Input Recommendations in the TruSeq Synthetic Long-Read DNA Library Prep Guide.
Follow these DNA input recommendations:
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.
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% RHStorage 5–95% RH
Illumina provides each customer with a seat license for Illumina GenomeStudio software. Illumina provides all samples and markers in a GenomeStudio project workspace. The Project Scientist zeros poorly performing samples and markers. These samples and markers remain available, enabling customers to make their own determination. Illumina provides genotyping data files, which indicate the bi-allelic genotyping call with each genotype on a separate row. The exported data files are customizable, and the Project Scientist asks customers for their preference before delivery. Optional data fields include intensity values, allelic strand formats, etc. The deliverables also include intensity files and locus and DNA summary files that show project statistics. Illumina delivers all data via a secure FTP site. The project scientist provides ongoing support for questions about the data.
The WGS analysis pipeline v3.0 uses Isaac Aligner, and Isaac Variant Caller to generate several outputs. These outputs include sequencing reads with reduced-resolution Q-scores in BAM format, and variant data in both VCF and genome VCF (gVCF)1 file format. The somatic small-variant calling component of the cancer analysis pipeline uses Isaac Aligner and Strelka2 to generate somatic small-variant data in VCF format. These informatics pipelines enable significantly increased alignment efficiencies and reduced data footprints, without compromising the quality of the data and variant calls.
A WGS sequencing analysis training package, a cancer analysis training package, and a human phasing training package are available in BaseSpace. A services user guideline and a quick video describing the deliverables are also available in BaseSpace. See the Sequencing Services Training Material for more information.
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.
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).
The Nextera Mate Pair Gel-Plus protocol with size-selection aims to generate mate pair libraries with a narrower size distribution and larger median fragment size. This is achieved by using a gel-based size-selection process to select DNA of a desired size range. A Mate Pair library with a narrow distribution of fragments will facilitate accurate structural variation detection. It will also help de novo assemblies of both simple and complex genomes by providing paired read information which spans larger repeat regions.
However, the degree of difficulty in generating gel-plus libraries increases as the length of fragments increases. Compared to libraries with smaller Mate Pair fragment sizes, libraries with larger fragment lengths are expected to have a lower final library yield and lower library diversity and are more challenging to make robustly. To increase the robustness of library generation when carrying out the Gel-Plus procedure, several steps in the protocol have been optimized and guidelines are provided.
The key advantage to using the TruSeq Nano DNA Library Prep kit is that only 100 ng of starting genomic DNA is required. This is increasingly important in studies that have limited amounts of DNA or where DNA needs to be split into multiple applications. In addition, the TruSeq Nano DNA Library Prep kit is a comprehensive solution containing all of the necessary reagents, barcodes, and size selection beads for convenient processing. The improved workflow includes bead-based size selection, therefore eliminating the time of gel-based methods. The kit also produces premier library quality, offering an excellent solution for studies requiring the highest coverage.
BaseSpace Variant Interpreter uses the following annotation sources: dbSNP, Catalogue of Somatic Mutations in Cancer (COSMIC), ClinVar, 1000 Genomes, Exome Variant Server (EVS), Exome Aggregation Consortium (ExAC), PolyPhen, and SIFT.
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.
Real-Time PCR has been used in quantification of gene expression, viral quantification, validation of array data, pathogen detection, and allelic discrimination.
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.
Refer to the TruSeq Library Prep Pooling Guide for recommendations and guidelines for Illumina sequencing systems that require balanced index combinations.
Sequencing libraries prepared with the TruSeq DNA PCR-Free Sample Prep Kit requires the HiSeq X Reagent Kit v2, or later. TruSeq DNA PCR-Free libraries are compatible only with workflow changes introduced with the v2 chemistry.
Reagent Kit Version
Control Software Version
TruSeq Nano DNA
Any HiSeq X kit version
Requires v3.0, or later
TruSeq DNA PCR-Free
Requires the HiSeq X v2 kit, or later
Requires v3.1, or later
The HiSeq X Reagent Kit v2.5 requires HiSeq X Control Software v3.1, or later.
The PC Module has the same computing requirements as other GenomeStudio Microarray modules listed here.
Coverage and read depth requirements vary considerably depending on the form of methylation sequencing used. Please refer to the following references for specific recommendations:
A typical MiSeq run produces FASTQ.gz files totaling 1–5Gb of sequence data.
Labs typically store zipped FASTQ files (*.fastq.gz) and Assign project files (*.cgp). The total size of all FASTQ files for a run mostly depends on the sequencer and the sequencing flow cell that was used and does not depend on the number of samples.
FASTQ File Size
FASTQ File Size Variance
Assign Project File Size
Assign Project File Size Variance
MiSeq v2 Nano (300 cycles)
+/- 200 MB
+/- 50 MB
MiSeq v2 Micro (300 cycles)
+/- 500 MB
+/- 50 MB
MiSeq v2 Micro (300 cycles)
+/- 500 MB
+/- 75 MB
MiSeq v2 (300 cycles)
+/- 2 GB
+/- 100 MB
MiSeq v2 (300 cycles)
+/- 2 GB
+/- 500 MB
|Sample Preparation||Quality DNA that has been chromatin immunoprecipitated with chemical modifications||Standard genomic DNA with no modifications|
|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|
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.
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.
For instrument specifications, see the Site Prep/Lab Environment page.
With the new, improved TruSeq Rapid Exome Enzyme, TDE2, the effects of adding a range of DNA is minimal. The protocol is optimized for 50 ng input DNA, and this amount is recommended. However, the protocol has been validated to have similar final analysis results from 30-75 ng DNA input.
A higher mass input of gDNA can result in incomplete tagmentation and larger insert sizes, which can impact enrichment performance. Conversely, a low mass input of gDNA or low quality gDNA in the tagmentation reaction can generate smaller than expected insert sizes, which can be lost during subsequent cleanup steps and result in lower diversity.
Overloading the Nextera tagmentation reaction with more than 50 ng of genomic DNA leads to a larger library size distribution, which can lower the number of on-target sequencing reads. Less than 50 ng of input DNA leads to a smaller library size distribution. The result is reduced library diversity or elevated duplicate reads because many of these smaller library sized fragments would be eliminated during the size-selection procedures.
Overloading the Rapid Capture reaction with too much library can lead to an increase in off-target reads. Conversely, loading too little library in the Rapid Capture reaction can lead to lower library diversity and an increase in duplicate sequencing reads.
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:
The yellow highlighted cells are genes that the panel has targeted.
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.
> 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.
The TruSeq DNA PCR-Free Library Prep Guide provides guidelines for both a 350 bp and 550 bp average insert size based on paired-end sequencing results. The 350 bp workflow starts with 1 µg of gDNA, and the 550 bp workflow starts with 2 µg of gDNA.
You should maintain a lab temperature of 22°C ±3°C.
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.
The controls that are required depend on the type of Real-Time PCR experiment:
The minimum system requirements are Windows 7 or later, 64-bit CPU, 2 GB RAM minimum, 4 GB recommended, and 25 MB hard drive space for installation.
Importing whole genome data or large gVCF files requires more RAM. For more information, see the entry in the File Formats section on this page, "What are the requirements for importing whole genome data or large gVCF files?".
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.
See the Cluster Densities Specifications technical bulletin on MyIllumina for recommendations.
Use the following minimum computing requirements:
Minimum Computing Requirements:
· 1 Ghz or faster 64-bit Intel core processor, or equivalent
· 16 GB RAM, minimum
· 16 GB available hard disk space
· Windows OS (Windows Vista, Windows 7, Windows 8, Windows Server 2008, or Windows Server 2012)
· Microsoft Excel 97, or later, for generating reports
|Loading Concentration (pM)||
Number of Libraries Per Flow Cell
Reads Per Sample (M)
MiSeq (v3 reagents)
The protocol has been developed and validated with 100 ng of high quality DNA for a 350 bp insert size and 200 ng of high quality DNA for a 550 bp insert size.
Importing large gVCF files requires more memory and takes longer to import. Use the following information as a guideline to estimate required RAM. This information is based on a Quad-Core Xeon® processor with 16 GB RAM.
Batch uploads from a network or local directory are limited to 100 files or 10 GB, whichever is greater. There are no limits on importing files from BaseSpace Sequence Hub.
For genotyping, Illumina requires at least 1.75 μg of pure and intact DNA per sample in 35 μl with a minimum concentration of 50 ng/ul as measured by PicoGreen. For methylation, Illumina requires at least 2.10 μg of pure and intact DNA per sample in 40 μl with a minimum concentration of 70 ng/ul as measured by PicoGreen. See the Illumina Genotyping Service Guidelines and the Illumina Methylation Service Guidelines for more information.
Illumina requires at least 2 μg of pure and intact DNA per sample. Refer to the Sequencing Service Process for more information.
Illumina Automation Control v5.3.0, Illumina LIMS v4.8.1, Tecan Tip Guide-E, Standard Teflow glass back plates and spacers, iScan Control Software v3.3.29, GenomeStudio 2011.1, BeadArray Controls Reporter.
There are 7 safe stopping points in the TruSeq Exome protocol. The safe stopping points are after the following steps: DNA Fragmentation, DNA Repair and Size Selection, Ligation, First PCR Amplification, First Elution, Second Elution, Final PCR Cleanup.
All are safely stored at -25° to -15°C for up to 7 days.
There are 4 safe stopping points in the workflow: (1) after long-range PCR, the amplicons can be held on the thermal cycler at 10°C, (2) after tagmentation clean-up at -20°C, (3) after index PCR at -20°C, and (4) after index PCR clean up at -20°C.
There are 7 safe stopping points in this protocol:
-After Synthesize Second Strand cDNA at -25°C to -15°C for up to 7 days
-After Ligate Adapters at -25°C to -15°C for up to 7 days
-After First PCR Amplification at -25°C to -15°C for up to 7 days
-After First Capture at -25°C to -15°C for up to 7 days
-After Second PCR Amplification at 2° to 8°C for up to 2 days
-After Second PCR Clean Up at -25°C to -15°C for up to 7 days
-After Clean up Captured Library at -25°C to -15°C for up to 7 days
For more information, see the reference guide.
There are 6 safe stopping points in the protocol:
-After First PCR Amplfication at 2° to 8°C for up to 2 days
-After First PCR Clean Up at -15° to -25°C for up to 14 days
-After First Capture at -15° to -25°C for up to 7 days
-After Capture Sample Cleanup at -15° to -25°C for up to 7 days
-After Second PCR Amplfication at 2° to 8°C for up to 2 days
-After Second PCR Clean Up at -15° to -25°C for up to 7 days
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.
Libraries prepared using the TruSeq Nano DNA Library Prep for NeoPrep Kit can be stored at -25°C to -15°C for up to 2 months.
Libraries prepared using the TruSeq Stranded mRNA NeoPrep Kit can be stored at -25°C to -15°C for up to 2 months.
The kit contains 3 boxes. Box 1 is stored at room temperature, Box 2 is stored at -25°C to -15°C, and Box 3 is stored at 2°C to 8°C.
The kit contains 3 boxes. Box 1 is stored at room temperature, Box 2 is stored at -25°C to -15°C, and Box 3 is stored at 2°C to 8°C.
Storage requirements for raw data are approximately 60% greater than current runs based on additional swath data and increased cluster density.
Sequencing an insert size of 550 bp is supported with NextSeq v2 reagents. With NextSeq v1 reagents, an insert size of 350 bp is supported.
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.
If you are upgrading from an earlier version of software (i.e., HCS 1.3 or lower), please contact Technical Support as you may need to re-validate. Validation of HCS 1.5/RTA 1.13 should not be required if you are upgrading from HCS 1.4/RTA 1.12. The changes in RTA do not affect data quality and the changes in HCS were to update the user interface to enable dual indexing. Please refer to the HCS 1.5 Release Notes for additional information about new features in this software package.
Typical output concentrations from the pre-enriched library are 40–125 ng/μl.
Typical output concentrations from the final enriched library are (nM calculation assumes a 400 bp library size):
Assign 2.0 first performs alignment of the sequencing reads. The alignment is performed against a locus consensus sequence generated from the alleles for each locus. The heterozygous positions of these aligned reads are then phased. The first pass of phasing phases heterozygous positions within the same read. The second pass of phasing phases heterozygous positions fulling within the same read pair. The final phasing step layers paired reads to determine phase between heterozygous positions for which the first 2 passes were unable to elucidate phase. If all 3 of these passes fail to determine phase or if the data provides ambiguous phasing, phase is not assigned and a phase break is shown.
These phased alignments are then compared to the IMGT/HLA database within Assign 2.0 and can be assigned an allele, multiple alleles, or no alleles. If an unambiguous alignment to a single allele is made, then this result is displayed on the summary report. If the result is ambiguous (multiple perfect matches), all perfect matches appear in the report. If no perfect match is available, these receive a no call and require manual review and editing, if warranted.
MiSeq Reporter can be viewed with the following web browsers: Firefox 13.0.1+, IE 11+, and Safari 5.1.7+.
Analysis can be performed using the TruSeq Amplicon BaseSpace App or MiSeq Reporter TruSeq Amplicon Workflow.
Perform analysis using the TruSeq Amplicon BaseSpace App or MiSeq Reporter TruSeq Amplicon Workflow.
The quality and quantity of genomic DNA input into the Nextera tagmentation reaction will affect the library size distribution. A larger peak distribution (> 350 bp) can be indicative of > 50 ng genomic DNA input going into the Nextera tagmentation reaction. Conversely, a smaller sample peak distribution (< 225 bp) can be indicative of < 50 ng genomic DNA or fragmented, low quality genomic DNA.
It is critical to accurately quantify the concentration of input genomic DNA. Illumina recommends quantifying the starting genomic DNA using a fluorometric-based method specific to double-stranded DNA, such as QuantiFluor or PicoGreen. For more information, see the DNA Input Recommendations section of the Nextera Rapid Capture Enrichment Guide.
The profile of the pre-enrichment library product can look different from the example shown depending on the type and quality of input DNA. Sometimes a larger molecular weight peak is present. This peak can be variable in size; however, it has minimal to no effect on the final exome metric output and you can proceed with the protocol. This peak is most often a result of the amplification step of the protocol.
The MiSeq System uses the MiSeq Reagent Kit, which includes specially designed and packaged reagents for cluster generation and sequencing.
For kit compatibility information, see the Illumina Version Compatibility Reference.
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.