Sequencing Questions & Answers

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  • Is there an optimal total number of tags per experiment in order to consider an experiment successful? What coverage is needed to confidently find peaks?
    Illumina cannot provide an optimal number because it depends on experimental design and antibody specificity. For example, specific transcription factors require a smaller number of tags than those that are more general (PolII, Histone, …). The higher throughput of the GAII system gives confidence that data from one or more lanes in a single flow cell may be sufficient for high confidence in the number of peaks found per region.
  • Are degraded or FFPE samples compatible with this protocol?

    The quality of DNA isolated from FFPE samples can be highly variable. Due to this variability, it is very difficult to reliably predict the quality of a library prepared from FFPE samples using 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.

  • Do I need to purchase AMPure beads if using the TruSeq DNA PCR-Free Library Prep kit?

    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.

  • Are there any protocol changes when using v3 reagents and flow cell v3?

    You need to increase your starting template concentration. However, the starting template volume (120 µl) is unchanged from previous kits.

  • How should I specify the splice junction set?

    As of CASAVA v1.7, eland_rna uses the refFlat.txt.gz or seq_gene.md.gz file to generate the splice junction set automatically.

  • What is the maximum recommended genome size for de novo assembly?
    The maximum recommended genome size for de novo assembly on the MiSeq is 20 Mb.
  • How many reads can I expect from an optimally clustered MiSeq flow cell?
    You can expect about 15 million reads passing filter.
  • Can I use a microheating system other than the one recommended for this protocol?

    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.

  • Does VariantStudio require an internet connection?

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

  • Can I import VCF files produced by non-Illumina software products into VariantStudio?

    Yes. However, if analysis software other than Illumina analysis software is used to generate data, the VCF file might not contain the columns required by VariantStudio. See the VariantStudio User Guide (part # 15040890 B) for information about VCF file requirements.

  • Can I use GenomeStudio to download data from the iControlDB database?
    No. Only the Illumina iControlDB-Client program can be used to download data from the database.
  • What is the difference between the open source version of the Isaac algorithms and the HiSeq Analysis Software? Which one should I use?

    The Open Source version of Isaac includes the component algorithms for the Isaac aligner and Variant Caller and is intended for developers. This version is not commercially supported and is provided as is under Illumina Open Source Software License available here.

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

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

  • The errorlog.txt file has many entries, such as Unable to copy Queued file (retry later), The network path was not found, or Logon failure: unknown user name or bad password. What should I do?

    During a typical run, Real Time Analysis (RTA) may occasionally run into copy issues, and will retry copying the file later. When the file is successfully copied, RTA proceeds normally. Therefore, seeing a few of these issues in the error log is acceptable. However, if you see many of these entries (dozens or hundreds), there could be a problem with your network. You might have an incorrect password , network path, or your network connection might not be fast enough. Try the following to troubleshoot this issue:

    1. Make sure that the network location is valid. Your network may be down, or a folder might have been moved.
    2. Make sure that you have the correct user name and password.
    3. Test whether your network is fast enough. See the FAQ “How can I check whether the copying of files proceeds fast enough?” for details.
  • What level of indexing is supported for TruSeq Small RNA Library Prep?

    TruSeq Small RNA Library Prep kits currently support 48-plex per lane.

  • Do I need to use a cBot to cluster a HiSeq/HiScanSQ flow cell?
    Yes. The HiSeq flow cell, also used on the HiScanSQ, requires the use of a cBot for clustering on the flow cell prior to sequencing.
  • When should you use the TruSight RNA Fusion Panel and when should you use TruSeq Targeted RNA Expression?

    TruSight RNA Fusion is a large gene panel covering 507 cancer-related genes. TruSeq Targeted RNA Expression typically provides smaller panels and fusion detection is more difficult. In addition, TruSight RNA Fusion has discovery power for fusion detection because only 1 gene fusion partner is required. With TruSeq Targeted RNA Expression, oligos must be designed either side of a known breakpoint. TruSight RNA Fusion is more amenable to low input FFPE.

  • Which Index Kit should I purchase if I will mainly be pooling less than 24 samples?
    Illumina recommends purchasing the 24 Index/96 sample Kit (Catalog # FC-121-1011) if your sample number is not greater than 24 for each library prep event. Based on the possible index combinations, in order to optimize the indices in the kit, the 24 Index Kit is recommended.
  • Are there different cBot kits for TruSeq HT dual-indexed libraries?

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

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

    HiSeq v4 SBS kits:

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

    TruSeq v3 SBS kits:

    • The 200-cycle kit contains reagents for 209 total cycles.
    • The 50-cycle kit contains reagents for 58 total cycles.

    HiSeq Rapid v2 SBS kits:

    • The 500-cycle kit contains reagents for 525 total cycles.
    • The 200-cycle kit contains reagents for 225 total cycles.
    • The 50-cycle kit contains reagents for 74 total cycles.

    TruSeq Rapid SBS kits:

    • The 200-cycle kit contains reagents for 225 total cycles.
    • The 50-cycle kit contains reagents for 74 total cycles.
  • What data quality can I expect from a run on the HiSeq 3000?

    You can expect greater than 75% of all bases above Q30 with a 2 x 150 bp run. For more information, see HiSeq 3000/HiSeq 4000 System Specifications.

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

    HiSeq v4 SBS kits:

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

    TruSeq v3 SBS kits:

    • The 200-cycle kit contains enough reagents for 209 cycles.
    • The 50-cycle kit contains enough reagents for 58 cycles.
  • What software is required for sequencing dual-indexed libraries?

    HiSeq users must upgrade to HCS1.5/RTA1.13 or higher and GA users must upgrade to SCS2.10/RTA1.13 or higher.

    Category

    Software Version

    Instruments

     Required

    Instrument Software and Sequencing Run QC

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

     

    HiSeq/HiScanSQ

    Yes

    SCS 2.10/RTA 1.13/SAV 1.8.20

    GA

    Yes

    Analysis

    CASAVA 1.8.2

     

    HiSeq/HiScanSQ/GA

    Yes

    Sample Sheet Creation

    Illumina Experiement Manager 1.3

    HiSeq/HiScanSQ/GA

    Recommended

  • What is a patterned flow cell?

    A patterned flow cell is a flow cell with billions of ordered nano-wells that are manufactured into the flow cell glass. The ordered wells allow for the generation of sequencing clusters in an ordered arrangement. Clusters are aligned more closely together increasing the number of output reads and amount of sequence data generated. The HiSeq X patterned flow cell contains 8 lanes and has the same general dimensions as a HiSeq high-output flow cell.

  • What is the shelf-life of the ForenSeq DNA Signature Prep Kit?

    Expiration dates are printed on the kit box labels and reagent tubes. Illumina tries to ensure 6 months of shelf life from time of kit shipment.

  • What if the values measured by qPCR and Qubit are very different during TruSeq Synthetic Long-Read DNA library prep?
    Qubit measurements are substantially higher because it measures total DNA. qPCR measures ligated, amplifiable fragments. It is important to accurately quantify the DNA by qPCR and use this value to calculate dilutions to make sure that the appropriate amount of DNA is used for Long Range PCR.
  • Why does this protocol use a thermal cycler instead of a microheating system for the Rapid Capture hybridization reactions?

    A thermal cycler leads to better consistency and more robust results across samples with a plate when compared to using a heat block for Rapid Capture hybridization. By using a thermal cycler at this step, the 100° heated lid helps to prevent large sample evaporation.

  • How will my TruSeq controls be shown for multiplexed samples?

    You can filter by Index in SAV (Sequencing Analysis Viewer) for indexed runs.

  • Is a wash required before starting a NeoPrep run?

    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.

  • Will CASAVA 1.8.2 be able to process both single-index and dual-index TruSeq runs?

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

  • How long does template generation take on the MiSeq?
    With MCS v2.3, template generation occurs during the first 7 cycles of sequencing. When using earlier versions of MCS, template generation occurs during the first 4 cycles of imaging.
  • Can I save .cif files in HiSeq v4 mode?

    In HiSeq v4 mode .cif files cannot be saved. The option to save .cif files is available in other modes.

  • What are the computing requirements for the PC Module?

    The PC Module has the same computing requirements as other GenomeStudio Microarray modules listed here.

  • When I started my run, I got the HCS error message, “Must have at least one valid ETF to normalize/correct the failed ones.” What does this mean and what should I do?

    This error message indicates a lack of fluorescence on the flow cell. To find focus at the start of a run, the software uses ETF, which is a focusing method that reads fluorescence from clusters on the flow cell. ETF must find fluorescence in at least one lane of the flow cell before the run can begin.

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

  • I received a warning message “need at least 750 GB space in local server” when starting a run. Is the run folder on the local computer 750 GB in size?

    This amount of free space is required at the beginning of a run. The system assumes that data 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.

  • Which Illumina sequencers are compatible with TruSight One libraries?
    TruSight One libraries can be run on all Illumina sequencing systems. A trio of TruSight One-enriched sample libraries can be sequenced so that >95% of the target regions are covered at 20x minimum coverage. This coverage can be achieved using the supplied 300-cycle MiSeq v3 reagent cartridge included as part of the 9-sample TruSight One kit. The 36-sample TruSight One kit does not contain sequencing reagents. Lack of reagents allows flexibility to prepare 12-plex pooled sample libraries that can be sequenced to target depths with a single flow cell using the HiSeq2500 in rapid run mode.
  • Do I quantify my final library from ForenSeq DNA Signature Prep?

    Final libraries are single stranded and cannot be quantified. Follow the normalization and pooling protocol before loading onto the MiSeq FGx.

  • Can I analyze rapid run data with CASAVA?

    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.

  • The Ambion TotalPrep™ kit is biased for eukaryotic genomes, whereas the microbial mRNAs lack poly-A tails. Does Illumina support any sample prep/labeling kit for microbial genomes?

    Illumina has not tested kits designed to label microbial RNA. Reagent vendors such as Ambion® do sell kits for this application. These kits may work, but there is no particular kit that we recommend. It is important that a single source of biotin-16-UTP (i.e., same vendor) is used for all labeling reactions (e.g., Ambion #8452 or #8453).

  • What are the workflow changes for sequencing on the MiSeq, HiSeq, and GA for TruSeq HT dual-indexed libraries compared to TruSeq v2 libraries?

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

  • Can I use the TruSeq DNA HT Sample Prep Kit to generate libraries from amplicons and FFPE samples or only from genomic DNA?

    All TruSeq DNA sample prep kits, including the HT version, supports input of standard genomic DNA only. Illumina offers other kits for Amplicon library prep. The TruSeq DNA HT Sample Prep kit supports high quality genomic DNA as input for sample prep to ensure best results. Please see the DNA Input Recommendations section of the TruSeq DNA Sample Preparation Guide for further information.

  • Can this protocol use purified small RNA as starting material?

    Yes. Please note that we have seen high sample variability using some commercially available small RNA spin column purification methods, and validation of any purification method may be necessary. If purified small RNA is used as starting material, 10–50 ng should be used.

  • The protocol recommends purifying 200 bp fragments from the gel. What if my fragments are not exactly 200 bps when run on a Bioanalyzer?
    While 200 bp is the ideal size for the Genome Analyzer, this means the insert will be about 100 bp because the combined flanking sequence (including adaptors) will be about 100 bp. This enables 35 bp to 50 bp single reads. NOTE: If the fragment is less than 150 bp, it might be difficult to obtain 50 bp reads.
  • I see a design gap in my Nextera Rapid Capture Custom Enrichment target region. Is there anything that I can do about this?
    Design gaps can occur due to complex genomic regions, low probe specificity, skewed GC content, and other technical factors inherent to probe based enrichment. The gap can be resubmitted for design as a New Target Region using the gap start and stop chromosome coordinates.
  • What is a TSO?
    TSO stands for TruSight Oligo, which is the final pool of oligonucleotide probes used to generate amplicons in the assay. A single TSO supports the amplification of all amplicons in a single reaction.
  • Can I use degraded total RNA for mRNA-Seq library production?

    While it is possible to create libraries for sequencing on the Genome Analyzer from degraded RNA, it will have a large effect on the performance of the assay. One of the first steps in the process is the purification of poly-A mRNA using a poly-T capture step. If the RNA is degraded, the mRNA that is captured at this step will not be full length, and will not give full-length cDNA products after random priming. High-quality total RNA produces even, end-to-end coverage of each mRNA molecule. If the RNA is degraded, you might observe a noticeable 3'- to 5'-bias in the number of reads for most transcripts. Greater degradation will result in greater bias.

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

  • How can I create subpanels for use in VariantStudio?
    Subpanels can be created by generating gene lists in HGNC nomenclature for the regions of interest. The complete TruSight One gene list can be found at (www.illumina.com/products/trusight-one-sequencing-panel.ilmn) for reference. Upload this subpanel gene list when importing *.vcf files into VariantStudio to filter variants based on the shortened gene list.
  • Can I import sample information (metadata) instead of entering it manually?

    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.

  • Because only a single strand is captured, are there a large number of incorrect methylation calls due to high frequency SNPs?

    Because bisulfite sequencing converts unmethylated cytosines to uracil, which eventually become thymines in the final sequence, in theory, C>T SNPs could be mistaken for “unmethylated” cytosines.  For regions containing CpGs with common SNPs (MAF >5% in EUR superpopulation 1000 genomes data), we designed probes to capture both strands. Thus, high frequency SNPs at CpGs which would be obscured if only a single strand was sequenced can now be called by looking at the second strand in these regions. If low frequency SNP calls (<5% MAF) at unmethylated CpG sites are desired, other suitable technologies are needed (eg, sequencing an unconverted sample).

  • Where can I find workflows for the GenomeStudio sequencing modules?
    You can find workflow documents for the GenomeStudio ChIP Sequencing, DNA Sequencing, and RNA Sequencing modules in the GenomeStudio Portal or on the Product Documentation web page.
  • What is the expected size of a MiSeq analysis folder?

    The size of the analysis output folder for each sequencing run depends on the number of cycles in your run. Typically, a 150-cycle paired-end run (2 x 151 cycles) generates approximately 3 GB in output.

  • When will Real-Time Analysis v2.7.7 be available to support lower PhiX spike in on HiSeq 3000/4000

    RTA Version 2.7.7 will be available to TruSeq Methyl Capture EPIC customers in Q4 of 2016.  It will then be released to all Illumina customers by 2017.

  • How many samples can I sequence at the same time with TruSeq Targeted RNA Expression?

    This is dependent on the experiment plexity and MiSeq version. Illumina recommends using the TruSeq Targeted RNA Calculator to determine the number of samples that should be pooled together to achieve the depth of coverage required for your assay. Refer to the TruSeq Targeted RNA Expression tech note for information on how to design an experiment and determine the appropriate number of samples to be run on a flow cell.

  • What are the compatibility requirements for using the HiSeq X Reagent Kit v2.5?

    The HiSeq X Reagent Kit v2.5 requires HiSeq X Control Software v3.1, or later.

  • What are the sizes of the TruSeq QC probes?
    There are 3 artificial dsDNA targets in the TruSeq Sample Prep Kit (CTE, CTA, CTL) that measure the enzymatic activities of each of the ERP, ATL, and LIG reagents. Each type of control probe ranges in size of 150-850 bp, in 100 bp increments. Illumina recommends selecting the 400-500 bp range library into cluster generation. However, for TruSeq Enrichment, Illumina recommends selecting a 300-400 bp range library.
  • What criteria determine clusters passing filter on Illumina sequencing systems?

    To remove the least reliable data from the analysis results, often derived from overlapping clusters, raw data are filtered to remove any reads that do not meet the overall quality as measured by the Illumina chastity filter. The chastity of a base call is calculated as the ratio of the brightest intensity divided by the sum of the brightest and second brightest intensities.

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

  • How many BeadChips can I scan at one time on the NextSeq 550 system?

    One BeadChip can be scanned at a time.

  • Should the RNA samples be DNase-treated?

    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.

  • Can I use a thermal cycler instead of the recommended microheating system?

    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. 

  • What flow cell should be used with ChIP-Seq libraries?
    ChIP-Seq libraries constructed with the ChIP-Seq sample preparation kit should be run on single-read flow cells. Libraries constructed with paired-end adapters can be run on either single-read or paired-end flow cells.
  • What flow cell should be used with ChIP-Seq libraries?
    ChIP-Seq libraries constructed with the ChIP-Seq sample preparation kit should be run on single-read flow cells. Libraries constructed with paired-end adapters can be run on either single-read or paired-end flow cells.
  • What is the minimum PhiX amount that can be spiked in?
    For most libraries, Illumina recommends using a low-concentration spike-in (1%) of PhiX Control v3 as an in-lane positive control for alignment calculations and quantification efficiency.
  • What is the amount of sequencing required on the various Illumina sequencers?
    The amount of coverage depends on the size of the sample genome and the type of sample. For more information, see the Coverage Calculation Tech Note and the Optimizing Coverage for Targeted Resequencing Tech Note.
  • What strand is sequenced in the TruSeq Stranded RNA sample prep kits?

    The cDNA strand is sequenced.

  • How do I prepare unconverted libraries using this kit?

    To prepare unconverted libraries (not used for methylation calls) with this kit, skip Bisulfite Conversion and go directly to the Amplify Enriched Library step.

  • Can TruSeq DNA and RNA libraries be run on older cluster kits?

    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.

  • Can TruSeq DNA and RNA libraries be run on older cluster kits?

    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.

  • Where do I find the index sequences to enter into my sample sheet?

    The Dual Indexing Principle section of the library prep reference guide contains a list of index sequences used in the kits.

    Alternately, Illumina Experiment Manager (IEM) software can be used to create your sample sheet and assign correct index sequences. Download the IEM software can be from the Downloads page.

  • Are there in-line controls included in the TruSeq Small RNA Library Prep Kit?

    There are no in-line controls for the TruSeq Small RNA Library Prep Kit.

  • Are there BED files available?

    Yes, download the BED files from Downloads page.

  • Why do some antibodies work for ChIP and others do not?
    ChIP assays require that the epitope recognized by the antibody be available/exposed after cross-linking and not obscured in the middle of a protein complex. Antibodies have to be of very high avidity (strength of multiple interactions) so that the interaction with the protein will survive the washing steps. It also requires that the protein you are trying to immunoprecipitate cross-links efficiently to the chromatin.
  • How does oligo design work for TruSeq Targeted RNA Expression? Can I design custom oligos to any gene of my choosing?
    The TruSeq Targeted RNA Expression assay consists of predesigned probes. Once a gene is selected, DesignStudio provides a list of predesigned probes. Due to the predesigned nature of these probes, DesignStudio design time is negligible. Options for probes include:
    • Transcript specific
    • 5' end
    • 3' end
    • cSNP
  • Do I need a mouse and keyboard for the NeoPrep System?

    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. 

  • What are best practices for reaching the right number of reads per sample?

    Accurate library quantification is needed to achieve even pooling for enrichment as well as the recommended raw cluster densities. Inaccurate quantitation and pooling can result in higher representation of some samples compared to others in the same pool. Accurate quantification of the final library before loading is needed to reach the recommended raw cluster densities.  Illumina recommends using the same diluted library for both quantitation as well as clustering.  If cluster densities are lower than expected, Illumina recommends checking the library size used in the concentration calculation using a Bioanalyzer trace and adjust accordingly.

  • What cluster generation kit is compatible?

    For kit compatibility information, see the Illumina Version Compatibility Reference.

  • How does Illumina define a validated assay?
    Assays marked validated in DesignStudio have been shown to give results consistent with fold changes observed by RNA-Seq in testing across human tissues.
  • How long are the upstream and downstream probe sequences?

    The length of the upstream and downstream probe design can vary between 22 bp and 30 bp, regardless of the size of the amplicons being designed.

  • What analysis solutions are available in BaseSpace Sequence Hub?

    RNA Seq-Alignment and TopHat Alignment are some analysis options on BaseSpace.

  • Can I use Sequencing Analysis Viewer (SAV) to view data from a HiSeq 3000 run?

    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.

  • What is the difference between the TruSeq Nano DNA LT and HT Library Prep kits?

    The TruSeq Nano DNA LT Library Prep kits come with single-index adapter tubes recommended for preparing 24 or less samples at a time. The LT kits come in two sets, A and B, and each set contains 12 unique single-index adapter tubes for a total of 24 unique single-index adapters if both kits A and B are combined. Each kit (LT kit A or LT kit B) come with reagents sufficient for 24 samples. The TruSeq Nano DNA HT Library Prep Kit comes in a 96 well adapter plate format with 96 dual-indexed adapters and enough reagents for 96 samples.

  • What is the minimum number of samples that can be prepared using this protocol?

    One sample can be prepared at a time. However:

    • When using the 8 rxn kits, it is most economical to process and pool 8 samples. However, pooling is not performed when using the 8 rxn × 1 plex kit.
    • When using the 12 plex or 288 sample kits, it is most economical to process and pool 12 samples for compatibility with the reagent kit configuration.
  • What kits are available and how many samples can be processed?

    You can purchase standalone library prep reagents or a kit that includes both the library prep and sequencing reagents.

    The following kits are available:
    Catalog # OP-101-1001: TruSight Tumor 15 MiSeq Kit (includes library prep and sequencing reagents), for 24 Samples (48 libraries) with 3x MiSeq v3 reagents (600 cycle)

    Catalog # 20005610: TruSight Tumor 15 MiniSeq Kit (Includes library prep and sequencing reagents), for 24 samples (48 libraries) with 3x MiniSeq High Output reagents (300 Cycle)

    Catalog # OP-101-1002: TruSight Tumor 15 (library prep only), for 24 Samples (48 libraries), no sequencing reagents

    Additional MiSeq reagent kit v3 (600 cycle) can be ordered with catalog # MS-102-3003.

    Additional MiniSeq High Output reagents (300 cycle) can be ordered with catalog # FC-420-1003.

  • Can I run my HiSeq system with different run modes on each side?

    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.

  • Is there a reason why my favorite gene is not included in the TruSeq Targeted RNA Expression predesigned probe list? Will it be added in the future?
    Every effort was made to include as many transcripts as possible. For human and mouse transcripts, refseq transcript models were used together with the hg19 and mm9 +mm10 genomes, respectively.
  • How are NeoPrep library concentrations reported?

    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.

  • How should a run be set up on a MiniSeq?

    If using Local Run Manager for on-board analysis on the MiniSeq, select the RNA Fusion module and accept the default sequencing parameters of 2 x 76 bp with single Truseq LT index. 

  • Does the NextSeq system perform data analysis on the instrument?

    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.

  • Can I process a low-diversity sample on a HiSeq 4000?

    True low diversity samples, such as single amplicon and 16S samples, are not expected to produce quality results. However, spiking in a well-balanced genome such as PhiX improves performance.

  • Is there an on-instrument analysis solution?

    No, there is no on-instrument analysis solution.

  • What reagents do I need to perform bisulfite sequencing applications?

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

  • How do I quantify the final libraries?

    A qPCR or fluorometric quantification assay using dsDNA binding dyes (such as Qubit or PicoGreen) can be used to quantify the final libraries.

  • What are the sample pooling guidelines for Nextera Enrichment libraries?
    The Nextera Enrichment DNA Sample Preparation kit supports pre-enrichment pooling of up to 12 different indexed samples. For pooling libraries prior to enrichment, it is recommended to pool libraries so all Index 1 (i7) indexes are unique. With this pooling approach, samples can be sequenced using a single index read workflow, as described in the relevant instrument User Guide. If Index1/i7 indexes are not unique, ensure that libraries with different Index2/i5 indexes are included (e.g. N703/E501 and N703/E502). With this approach, samples can be sequenced using a dual index read workflow, as described in the relevant instrument User Guide. For further details regarding sample pooling, please refer to the “Library Pooling Guidelines” section in the Nextera Enrichment Sample Prep Guide.
  • What software is required/available to support Nextera dual-indexing? Which versions?

    Software Component/version:

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

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

  • What controls should I use for sequencing low-diversity samples on a MiSeq?
    Illumina recommends adding PhiX at 1% to all libraries as an internal control. For low-diversity libraries, the percentage of PhiX depends on the diversity of the library and requires optimization. Monotemplates may require at least 30% PhiX. If you are using RTA 1.17.28 (released with the MiSeq v2.2 updater) or later, low-diversity libraries requires a minimum of 5% PhiX spike-in. A PhiX control is not required for libraries prepared with the TruSeq Custom Amplicon Kit or the Nextera XT kit.
  • Can I process a sample with low sequence diversity?

    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.

  • Which systems are eligible for the HiSeq v4 upgrade?

    System

    Serial Number

    HiSeq 2500

    SN# D00101 or higher

    HiSeq 1500

    SN# C00101 or higher

    HiSeq 2000

    SN# 7001403 or higher

    Field-upgraded HiSeq 2500

    SN# 7001403 or higher

    Field-upgraded HiSeq 1500

    SN# L179 or higher

    For more information, see the upgrade resources on the Overview tab.
  • What is the length of the oligo baits used in TruSeq Rapid Exome reactions?

    The Coding Exome (45 Mb) uses 80 mer oligos.  

  • What can cause my TruSeq Rapid Exome library to look different than the example shown in the TruSeq Rapid Exome Library Prep Reference 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.

  • I broke the gel when opening it during TruSeq Synthetic Long-Read DNA library prep. Can I still use it?
    Yes, as long as you are still able to accurately determine the position of the 10 kb and 8 kb bands of the ladder in relation to the sample.
  • How many samples can I prepare at the same time with TruSeq Targeted RNA Expression?
    Illumina recommends preparation of 16-96 samples at a time. Smaller batch sizes are supported, as long as reagents are not freeze-thawed more than five times. Larger batch sizes may be performed by more experienced users, taking care that the OB1 beads do not dry during purification steps.
  • What is the expected percentage of remaining rRNA after TruSeq Stranded Total RNA Ribo-Zero treatment?
    For standard (non-FFPE) samples less than 6% of ribosomal RNA contamination is expected. For FFPE samples, less than 6% is expected. These values vary depending on tissue quality and source.
  • How many cycles of sequencing are supported on the NextSeq system?

    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.

  • What is the Recurrent Fusions Not Called table?

    Recurrent Fusions Not Called is a table that displays recurrent fusions according to the Mitelman database where a fusion was not detected.  This table provides information to help the user assess if genes that are involved in recurrent fusions, where the fusion was not called, had sufficient read coverage. 

  • Are the Illumina adapter sequences available?

    Illumina adapter sequences are provided in the Illumina Customer Sequence Letter.

  • When will quality scores appear during a sequencing run on the MiSeq?
    Quality scores appear after cycle 25. The first 25 cycles are used to determine the chastity of a base call, which in turn determines the quality filter.
  • How many samples can be sequenced at a time?

    This kit has integrated sample barcodes that enable pooling of up to 96 samples per sequencing run. 

  • Can I save my filter settings as a template for use with other data sets?

    Yes. Any filter combination can be saved and applied to other data sets. For more information, see the BaseSpace Variant Interpreter Beta Online Help.

  • Why are the polymerase and FFNs missing from the HiSeq SBS Kit v4?
    The HiSeq v4 chemistry includes a pre-mixed incorporation reagent similar to the TruSeq Rapid kit.
  • What applications have been tested with ForenSeq DNA Signature Prep?

    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.

  • How long does the resynthesis step take on a HiSeq?
    The resynthesis step takes approximately 3 hours.
  • How many samples can I process using the TruSeq Exome protocol at 1 time?

    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.

  • What are the network requirements for data transfer from the HiSeq to a server?
    You need a one gigabit connection per instrument between the instrument computer and the server. For more information, see the HiSeq System Site Preparation Guide.
  • Can secondary analysis run on MiSeq while a run is in progress?
    If a new sequencing run is started on the MiSeq before secondary analysis of a previous run is complete, secondary analysis will be stopped automatically. MiSeq computing resources are dedicated to either sequencing or analysis, and the system is designed in such a way that a sequencing command overrides an analysis command. Secondary analysis can later be requeued from the MiSeq Reporter Analyses tab.
  • How frequently do you update your annotation database?

    Illumina does not have a specific schedule for updating the annotation database. This process largely depends on the cadence of updates from the different sources aggregated to create the annotation database.

  • Which workflow is required to create a TruSeq RNA Access Library Prep sample sheet using the Illumina Experiment Manager?
    • To create a HiSeq-compatible sample sheet, select the "HiSeq FASTQ Only" workflow and the "TruSeq LT" Sample Prep Kit.
    • To create a MiSeq-compatible sample sheet, select the "FASTQ Only" workflow and the "TruSeq LT" Sample Prep Kit.
  • Where do I load my samples on the cBot if I am using the HiSeq or TruSeq Rapid Duo cBot Sample loading kit?

    Load 135 μl of the denatured and HT1 diluted library into wells 1 and 2 of an 8-tube strip. Then load the 8-tube strip into the cBot tube strip holder, making sure to position wells 1 and 2 towards the right side.

  • What is the correlation between the Infinium Human Methylation EPIC BeadChip and TruSeq Methyl Capture EPIC?

    A Pearson correlation value of 0.96 was obtained.  For more information, see the TruSeq Methyl Capture EPIC data sheet.

  • Can the MiSeq Reporter software be run on a separate PC?
    MiSeq Reporter can be run on a 64-bit PC with at least 8GB RAM (16–32 GB RAM for optimal performance), Windows Vista or Windows 7, and at least 1 TB of available hard disk space. This allows secondary analysis to be performed offline.
  • What probe pool is used in the TruSight RNA Fusion Panel?

    The probes are specifically designed to target 507 fusion-associated genes covering all coding exons of each targeted gene.

  • How is Target Region Coverage calculated in DesignStudio?

    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.

  • Is amplicon contamination a concern with the MiSeq FGx System?

    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.

  • Which Illumina sequencing instruments are compatible TruSeq Targeted RNA Expression?
    TruSeq Targeted RNA Expression is currently supported on the MiSeq. MiSeq Reporter supports the protocol with its on-instrument analysis workflow.
  • Can the number of PCR cycles be reduced with the TruSeq ChIP Sample Prep protocol?

    The protocol is optimized for 18 cycles of PCR to allow for variability in sample input amounts and quality. High quality samples will likely be able to use a lower number of PCR cycles and still generated adequate amounts of final library. A titration of PCR cycles can be performed to identify the optimal performance for particular samples. Illumina does not recommending increasing the number of PCR cycles as it may skew the representation of the library.

  • Can I add custom genomic content to Illumina predesigned enrichment panels with Nextera Rapid Capture Custom enrichment?
    Yes, the add-on workflow in DesignStudio for Nextera Rapid Capture Enrichment allows you to add 350–67,000 custom probes to Illumina pre-designed enrichment panels (approximately 80 Kb–15 Mb of custom genomic content). Get started with designing your project in DesignStudio.
  • What is Local Run Manager?

    Local Run Manager is on-instrument analysis software integrated with the system control software. Using Local Run Manager, you can record sample information and specify run parameters before starting the run. The name assigned to the run appears on the control software run setup screen for faster run setup.

    When the sequencing run is complete, data analysis begins on-instrument automatically and performs analysis according to the analysis module specified for the run.

  • Is there an option to avoid indels in the design process?

    Yes. The ‘Avoid SNPs’ design option also avoids indels from the given variant database being used when possible (eg dbSNP for human designs).

    For important regions with known indels, Illumina recommends inspecting the probes using the linked UCSC tracks per item in the grid. In certain cases, probes overlapping indels can be the best or only option to provide coverage of that region.

  • How are the HiSeq X reagent kits configured?

    Each kit includes two flow cells and all required reagents, including clustering, SBS, paired-end, and indexing reagents. One kit provides consumables for a 150-cycle paired-end (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.

  • What can I use to analyze my data?

    Perform analysis using the TruSeq Amplicon BaseSpace App or MiSeq Reporter TruSeq Amplicon Workflow.

  • How do I set up runs so that secondary analysis is not performed on instrument but is performed solely by MiSeq Reporter running on another computer?
    To skip analysis (everything after FASTQ generation), you can specify the GenerateFASTQ workflow in the sample sheet. On the external computer, set the repository location using the Settings window on the MiSeq Reporter interface or edit MiSeq Reporter.exe.config file. The repository is the folder containing data files ready for analysis.
  • Can I use Sequencing Analysis Viewer (SAV) to view data from a HiSeq X run?

    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.

  • What should I do if there is an issue with the Seq part of my ChIP-Seq?
    Please refer to the standard protocols for the resequencing of genomic DNA.
  • What should I do if there is an issue with the Seq part of my ChIP-Seq?
    Please refer to the standard protocols for the resequencing of genomic DNA.
  • How do you make sure that different adapters are ligated to each end of a DNA fragment using this protocol?

    Illumina’s proprietary method ensures ligation of 2 different adapters in the required orientation to opposing ends of a DNA fragment. PCR selects for these and finalizes the construct ready for hybridizing onto the flow cell surface. Adapter sequences can be determined by sequencing the ligation fragments, but sequence information alone is not sufficient to uncover the method.

  • Which barcode scanner do I use to record consumable IDs?

    For runs with sample tracking, consumables are scanned internally after they are loaded onto the instrument and the lid is closed. Use the exteranl scanner to record consumable IDs for runs without sample tracking.

    If a consumable cannot be scanned internally, the software provides options for using the external scanner or onscreen keyboard. Sample tracking is not available for consumables that are not scanned internally, but the barcode IDs are always stored in the run data file, regardless of sample tracking.

  • How do I analyze my data using third party software?

    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, such as Bismark and MethylKit.

    Illumina cannot provide support for the use of third party software.  Contact the software resources directly with any questions regarding the analysis and use of the software. 

  • Are there any in-line sample prep controls in the TruSeq ChIP Sample Prep kit?

    The TruSeq ChIP Sample Prep kit does not contain in-line controls due to the low input amount.

  • Is quantitation performed before or after pooling of the libraries during this TruSeq Sample Prep?
    Samples should be quantified prior to pooling. It is possible to quantify after pooling if all DNA samples are of similar quality, but this requires very consistent yields and should not be attempted by a new user. See the appropriate TruSeq DNA or RNA sample preparation guide for details.
  • Do I get a single long-read from each TruSeq Synthetic Long-Read DNA barcode (384-well) or are there multiple synthetic long reads resulting from a single well?

    On average, there are several hundred long-read molecules per well. Individual wells produce many long read fragments randomly sampled from across the genome.

  • Are there third-party software programs that you can recommend for ChIP-Seq analysis?
    There are a number of solutions available, including Bioconductor and MACS, which are available through Galaxy.
  • Can I import custom annotations?

    Yes. You can import custom annotations to the software using the Custom Annotation function in VariantStudio. This feature requires that a custom annotation file uses a tab-delimited text format (TSV) and contains a header row that specifies the column names. VariantStudio allows both custom variant annotations and custom gene annotations.

  • Can I save images from a run on the HiSeq or HiScanSQ?
    No. Images are deleted automatically after they have been processed.
  • Can I change the default number of PCR cycles and other parameters for the NeoPrep library prep process?

    Some options can be modified and the options vary by assay. For more information, see the appropriate library prep reference guide.

  • Can I expect changes when using Sequence Analysis Viewer (SAV) with MCS v2.3 and MiSeq v3 reagents?

    Intensity (Data by cycle) plots appear different due to non-linear exposure ramping. Non-linear ramping prevents exposure damage early in the read, which provides a boost later in the read when it is more necessary.

  • How long can rapid SBS reagents be stored after initial preparation?

    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.

  • Can I perform sample to sample comparisons?

    No, BaseSpace Variant Interpreter does not currently support direct comparison of analysis results from 2 different samples.

  • Can I transfer called SNPs from CASAVA to use in custom BeadChip design?
    Called SNPs from CASAVA can be parsed to ADT via two reports: a dbSNP Report for the start and stop positions of the region flanking the SNP, and a Sequence Report which provides the actual sequence of the region flanking the SNP.
  • Can I transfer called SNPs from CASAVA to use in custom BeadChip design?
    Called SNPs from CASAVA can be parsed to ADT via two reports: a dbSNP Report for the start and stop positions of the region flanking the SNP, and a Sequence Report which provides the actual sequence of the region flanking the SNP.
  • How do I check the quality of my TruSeq Exome library?

    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.

  • How much additional time will be added to the current sequencing run time on HiSeq to support dual indexing?
    When using v3 SBS reagents and the v3 flow cell on HiSeq, each additional index cycle is approximately 53 minutes per cycle (with 16 total indexing cycles) plus ~2 hours for the seven chemistry-only cycles for the PE workflow, resulting in ~16 hours of additional time for dual indexing on HiSeq.
  • Can I use LCM-derived total-RNA?
    While this might be possible with as little as 100 ng with high-quality total RNA, Illumina does not have direct experience with this method.
  • Are custom libraries supported on the NovaSeq System?

    Yes. The NovaSeq Sequencing System permitts custom libraries. However, the NovaSeq denature and dilute protocols might require further titration and optimization to achieve optimal results for custom libraries.

  • What software should be used for analysis of TruSeq stranded mRNA libraries?
    Demultiplexing of dual-indexed sequencing runs requires CASAVA1.8.2. Alignment of single read runs may be performed using Casava 1.8.2. For alignment of paired-end runs, Illumina recommends TopHat/Cufflinks. For more information,  see the RNA Sequencing Analysis With TopHat guide. TopHat is not an Illumina supported product, but an open source initiative. Additional, third party analysis tools are also available. Learn more about Illumina's RNA applications.
  • Can TruSeq LT Set B indexes be used to increase index pooling options?

    TruSeq LT Set B indexes have not been validated with this workflow and therefore is not supported by Illumina.  

  • MiSeq Reporter indicates that I have a sample numbered 0. What is sample 0?
    Sample 0 is not designated in the samplesheet. Reads that were not successfully assigned to a sample are written to a FASTQ file for sample number 0, and excluded from downstream analysis.
  • Does the install of the PC Module require a license key?
    No, the PC module is a standalone software which does not require a license key.
  • Is the TruSeq DNA PCR-Free Library Prep kit suitable for GC-rich samples?

    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.

  • What types of target sequence inputs are accepted in DesignStudio when creating a project?

    The following inputs are accepted in DesignStudio:

    1. Chromosome number with location of starts and stops
    2. Gene name (with an option for exons only as well as up- and down-stream bases)
    3. Batch upload with a *.csv file. 

    Templates for coordinates and genes are provided in the file upload section for adding new target regions. For additional information, see the DesignStudio online help.

  • How many cycles do MiSeq v3 kits support?

    MiSeq v3 kits are kitted for 150 cycles and 600 cycles.

  • What’s new in NextSeq Control Software (NCS) v2.0?

    NCS v2.0 adds support for scanning BeadChip workflows. Scanning BeadChips on a NextSeq 500 system requires a hardware change. 

  • What is the maximum sized genome I can sequence on a MiSeq?

    For best results, Illumina recommends small genome (up to 20 Mb) sequencing on the MiSeq.

  • What methods of shearing are supported by TruSeq DNA Sample Prep?

    Illumina recommends Covaris shearing, but testing with nebulization shows comparable data.

  • Can I continue to use the NextSeq 500 Kit (v1) after upgrading to NCS v1.4?

    NCS v1.4 is not compatible with the NextSeq 500 Kit (v1). Use NCS v1.3, or earlier, with v1 kits.

  • What consumables and equipment do I need?

    A full list of user supplied items can be found in the Consumables and Equipment section of the library prep reference guide.

  • Can I use Sequencing Analysis Viewer (SAV) to view sequencing data from a NextSeq system?

    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.

  • If I reannotate my samples from the VariantStudio v2.1 projects in VariantStudio v2.2, can I keep both sets of annotations?

    No. If samples are reannotated in VariantStudio v2.2, all annotations for variants are replaced with the annotations from the newer version of  the Illumina Annotation Service. If you do not want to lose previous annotations for these samples, create a project for the samples that you want to preserve with the old annotations.

  • How do I requeue a run for analysis in MiSeq Reporter?

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

  • Which cluster kits are compatible with these libraries?

    All paired-end cluster kits are compatible with TruSeq Rapid Exome libraries. If preparing these libraries for cluster generation for HiSeq, HiScanSQ, or GAIIx, use the TruSeq Dual Index Sequencing Primer kit (catalog # PE-121-1003). Use the Read 1 sequencing primer (HP10) in place of the existing Read 1 sequencing primer (HP6) in the cBot plate. For more information on using HP10 in the cluster generation process, see the Preparing HP10 for Dual-Indexed Nextera Libraries section of the cBot User Guide.

  • What is the difference between the v1.5 small RNA and TruSeq small RNA sample preparation kits?
    1. The TruSeq kits allow indexing of up to 48 samples per lane. The v1.5 kits do not support indexing.
    2. The TruSeq kits have been optimized to simplify the workflow and reduce adapter dimer formation.
    3. The TruSeq kits allow paired-end sequencing, useful for directional RNA sequencing.
  • Can I pre-load samples, reagents, and oil on the NeoPrep library card before placing it on the stage?

    No, load the library card when it is on the stage to avoid spilling oil or disturbing the contents after loading.

  • What index sequences are used?

    This protocol uses TruSeq LT Set A indexes. The indexes can be found in the reference guide.

  • Is a uracil-tolerant PCR polymerase supplied with the kit?

    Uracil-tolerant PCR Polymerase is not supplied. However, purchasing Kapa HiFi HotStart Uracil + ReadyMix (2x) from Kapa Biosystems is recommended. Illumina has tested seven different commercially-available uracil-tolerant polymerases, and only Kapa Hifi gave results within Illumina specifications.

  • How does Isaac improve the speed of analysis 4–6 times over existing methods?

    The Isaac aligner aligns reads by first identifying a small but complete set of relevant candidate mapping positions. The Isaac aligner begins with a seed-based search, using 32-mers as seeds. The initial single-seed search is followed by a multi-seed only for the reads that couldn't be placed unambiguously with a single seed. Speed up is achieved by sorting the reference index by the 32-mers.  Improvement to accuracy is achieved by flagging of all the ambiguous reference positions in the index.

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

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

  • How do I designate dual indices on a TruSeq sample sheet?

    Designating dual indices on the sample sheet depends on whether a MiSeq or CASAVA sample sheet will be used. For MiSeq sample sheets, each index is entered into its own column. For CASAVA sample sheets, the indices are input in the format of "Index1-Index2". Please see the MiSeq Sample Sheet Quick Reference Guide or CASAVA User Guide for more information.

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

  • What are the longest Indels that Isaac has been able to detect reliably?
    10bp continuous indel over the length of the read.
  • What additional functionality does cBot 2 offer?

    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.

  • What is a tile?
    A tile is an image captured by the camera on the Genome Analyzer. A flow cell contains eight lanes. Each lane is imaged in two columns with 60 tiles from each column.
  • Is a Bioanalyzer trace required to be run after enrichment to check the library size?
    A post-Enrichment Bioanalyzer quality control step is optional. Running a Bioanalyzer trace can be a useful tool for troubleshooting lower than expected cluster densities by recalculating the post-Enrichment library concentrations with the returned library size estimate. If the average post-Enrichment library size is larger than expected, the actual loading concentration is lower than expected, resulting in lower raw cluster densities.
  • What do I do if I want to run fewer cycles than 50 or fewer cycles than 300?
    Use one of the available reagent kits that covers up to the number of cycles you want to perform. Any unused reagents are automatically discarded at the end of the run.
  • What software can be used to analyze TruSight RNA Fusion libraries?

    MiniSeq: The on-instrument Local Run Manager RNA Fusion Module performs alignment with STAR and analyzes for gene fusions with Manta.  The Local Run Manager RNA Fusion Module generates a summary report of fusions. 

    MiSeq: The off-instrument Local Run Manager and the RNA Fusion Module performs alignment with STAR and analyzes for gene fusions with Manta. The Local Run Manager RNA Fusion Module generates a summary report of fusions. Install the off-instrument Local Run Manager on a compatible PC first, and then install the RNA Fusion Module.

    BaseSpace: The BaseSpace RNA-Seq Alignment App performs alignment and analyzes for gene fusions with STAR/Manta or TopHat. STAR/Manta is recommended for optimal fusion calling. When STAR is selected as the aligner in the BaseSpace RNA-Seq Alignment App then Manta is used as the fusion caller.

    The BaseSpace TopHat Alignment App can also be used for alignment and fusion calling. The BaseSpace Cufflinks Assembly & DE App can be used to run differential expression analysis.

    Third party analysis tools are also available, including open source Manta and STAR.

  • Can I calculate PC and PPC errors in the PC Module?

    No, this is not an option in the PC Module.

  • What if UCSC doesn'€™t have a reference genome for the species I am interested in?

    The genome files for non-human species conform to the UCSC format. The GenomeStudio 2008.1 Framework User Guide, available in iCom and the GenomeStudio Portal, describes how to create genome files for non-UCSC genomes. Illumina is currently working on tools that will convert NCBI formats to UCSC format. Check the GenomeStudio Portal for updates.

  • What is a CAT?

    CAT stands for Custom Amplicon oligo Tube, which is the final pool of oligonucleotide probes used to generate amplicons in the assay. A single CAT supports the amplification of up to 1,536 amplicons in a single reaction.

  • The CASAVA calls SNPs at areas where the coverage dips.
    These SNPs may not be real SNPs, but small indels. A small indel will cause a short run of snp calls (~indel+4) with a concomitant dip in coverage. Check whether the apparent SNP can be explained by a short indel.
  • What are the highlighted cells in the Fusion Calls table?

    The yellow highlighted cells are genes that the panel has targeted.

  • In what format does MiSeq Reporter output aligned data?
    MiSeq Reporter outputs aligned data in the BAM file format.
  • What is the estimated success for sequencing custom targets with the Nextera Rapid Capture Custom Enrichment workflow?

    While coverage depth of individual target regions may vary, all targeted regions should be represented in the final enriched library.

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

  • Do I need a dedicated server to analyze run data from the NextSeq system?

    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.

  • How can I get a full list of the genes covered by the TruSeq Amplicon - Cancer Panel?

    The manifest file is available for download from MyIllumina.

  • Can the microTUBE AFA Fiber Screw-Cap be used to fragment samples on the Covaris M220?

    Illumina does not recommend using the microTUBE AFA Fiber Screw-Cap tube (Covaris Part # 520096) with the TruSeq Nano DNA 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).

  • What peripheral equipment is needed to perform the Illumina Nextera sample prep?
    A standard thermocycler is required to perform PCR.
  • Does this protocol offer strand-specific information?
    This protocol is designed for quantitative shotgun cDNA sequencing. Therefore, you get reads from both ends of the cDNA fragments and not from a specific strand. We determined that this protocol is the best method available for creating even, unbiased coverage of the entire length of the cDNA molecule. Our scientists tested many methods and found that this feature of the protocol provides the most quantitative and reproducible results for counting transcripts. If you are interested in generating strand-specific information, several publications have demonstrated this capability on the Genome Analyzer: for example, Lister et al., Highly Integrated Single-Base Resolution Maps of the Epigenome in Arabidopsis, Cell (2008), doi:10.1016/j.cell.2008.03.029.
  • How many samples can be sequenced at the same time when performing the TruSight DNA Amplicon assay?
    The TruSight DNA Amplicon has integrated sample barcodes that enable pooling of up to 96 samples per MiSeq run. However, the actual number of samples that should be pooled together per sequencing run depends on the number of amplicons and the desired depth of sequencing coverage. For TruSight Myeloid libraries, eight samples per MiSeq run with MiSeq v3 reagents should give the appropriate amplicon sequencing depth.
  • What is the difference between the TruSeq DNA PCR-Free library prep HS and LS protocols?

    The TruSeq DNA PCR-Free Library Prep Guide contains both a Low Sample (LS) and High Sample (HS) protocol. These protocols differ in the types of plates used and the method of incubation and mixing. The LS protocol uses 0.3 ml PCR plates, the incubation steps are done on a thermal cycler, and mixing method is pipetting. The HS protocol is done in MIDI plates, requires additional equipment such as microheating system for all incubation steps, and mixing method is done on a microplate shaker. The LS protocol is optimized for processing 24 samples at a time and HS protocol for more than 24 samples. Both the LT and HT kits can be used with either the LS or HS protocol.

  • What percent rRNA is expected?

    The expected percentage of passing filter reads aligned to human ribosomal RNA is typically ≤ 8%.

  • What additional equipment do I need with a MiSeq system?

    The MiSeq includes all the hardware needed for cluster generation, sequencing, and data analysis. More advanced analysis requires additional computing infrastructure. Other equipment may vary with application and sample prep methods, which is outlined in sample prep documentation.

  • What criteria were used for the design of human cSNP assays and what database was used for TruSeq Targeted RNA Expression?
    Human dbSNP 135 was used for the designs and the following criteria were considered for cSNP assays:
    • cSNP must be common (i.e., those with ≥ 1% MAF have been included)
    • There should be no other cSNP in the probe design area
    • Only 1 SNP per target is allowable
    • cSNP must be covered in all transcripts to a gene
  • How long does the Nextera Enrichment sample prep take?
    Samples can be prepared in less than 3 hours and complete enrichment of up to 96 samples can be completed in 2.5 days.
  • Which flow cell can I use to perform an instrument wash on my HiSeq system?

    Any previously used flow cell that has been stored properly in buffer can be used for the instrument wash. It is easiest to use the flow cell from the recently completed run to perform post-run and maintenance washes. Both v3 and v4 flow cells can be used to wash after high output modes. After Rapid Run mode, both HiSeq v2 and TruSeq flow cells can be used.

  • How does this kit differ from the TruSeq Stranded Total RNA and TruSeq Stranded mRNA library prep kits?

    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.

  • How am I notified when there is a new version of the Illumina Annotation Service?

    If a new version of the Illumina Annotation Service is released at the same time as a new software version release, the updates to the annotation database are specified in the software release notes. Otherwise, you receive an email notification informing you that a new database version is available. The email outlines the changes made relative to the previous annotation database version.

  • What software versions are required to sequence my prepared libraries?
    • IEM: Illumina Experiment Manager 1.5 software or later
    • cBot software (if needed): cBot 1.4 software or later
    • HCS software for HiSeq/HiScanSQ: HCS 1.5/RTA 1.13 software or later
    • SCS software for Genome Analyzer: Genome Analyzer SCS 2.10/RTA 1.13 software or later
    • HiSeq Analysis Software (HAS): any version
    • MiSeq Control Software (MCS): 2.2 software or later
    • MiSeq Reporter Software (MSR): 2.2 software or later
  • Does the Library QC workflow in MiSeq Reporter perform alignment?
    Yes, alignment is performed in the Library QC workflow; however, it is performed using a faster and less sensitive setting, which provides a much faster turnaround time. Variant calling is not employed for this workflow.
  • Is there sample data I can view?

    Datasets are available on the BaseSpace Public Data page.

  • How do I know that MiSeq Reporter has completed its analysis?
    When MiSeq Reporter analysis is finished, a checkmark appears in the State column of the Analyses tab and the CompletedJobInfo.xml file is written to the root level of the analysis folder.
  • How many samples can be processed per TruSeq DNA PCR-Free LT and HT Library Prep Kit?

    The TruSeq DNA PCR-Free LT kits contain sufficient reagents for 24 samples and the TruSeq DNA PCR-Free HT kit contains reagents for 96 samples. Illumina recommends using the LT kit if processing less than 24 samples at a time and the HT kit if processing more than 24 samples. Both the LT kit and HT kit can be used with either the Low-Sample (LS) or High-Sample (HS) protocols.

  • What are the coverage, read depth requirements, and sample input amounts for sequencing-based methylation applications?

    Coverage and read depth requirements vary considerably depending on the form of methylation sequencing used. Please refer to the following references for specific recommendations:

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

       

  • What is a PhiX spike-in?
    A PhiX spike-in employs a small amount of PhiX control in the same lane as a sample. This allows real time quality metrics as the PhiX is analyzed during the run. This is not recommended for sequencing a genome with high similarity to the PhiX genome, and does not allow for normalization of data in that lane as per a control lane.
  • What sequencing primers are needed?

    Standard sequencing primers included in the cluster generation kits are required.

  • Can Covaris settings be altered to produce fragments of different lengths?

    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.

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

    Samples

    Enrichment Reactions

    Plexity

    8

    8

    1

    24

    8

    3

    48

    8

    6

    72

    8

    9

    24

    2

    12

    48

    4

    12

    96

    8

    12

    288

    24

    12

  • Where can I find the manifest?

    The manifest can be found in the download tab of the product support page.

  • What is the maximum number of rows allowable in the target region file input method in DesignStudio?

    The maximum number of rows allowable is 1000. You can submit multiple files per project.

  • Why is there more volume in my sample tube at the end of my rapid run than when I started?

    At the end of all rapid sequencing runs, the instrument flushes water back into the sample tube to clean out the lines and prevent drying. This flush is an automatic procedure and does not require user intervention. This procedure dilutes sample left in the tube. Therefore, any sample left in the tube should be discarded after the run.

  • Are FFPE samples supported?

    Only use FFPE-derived DNA 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 library prep with FFPE samples, use the Illumina TruSeq FFPE DNA Library Prep QC Kit to determine input amount based on ∆Cq.

  • How do I analyze my TruSeq Targeted RNA Expression data?
    Use MiSeq Reporter for on-instrument analysis. This software provides analysis of replicates and allows for pair-wise comparison of targeted regions.
  • What are the effects of putting too much or too little tagmented library into the enrichment Rapid Capture reaction?

    Overloading the Rapid Capture reaction with too much library can lead to an increase in off-target reads. Conversely, loading too little library in the Rapid Capture reaction can lead to lower library diversity and an increase in duplicate sequencing reads.

  • Are there considerations for combining certain TruSeq Small RNA indexes or only combining 1-2 indexes?

    Testing has shown that performance shows minimal variability when combining low numbers of indexes, or across particular combinations of indexes, so any number or combination may be used.

  • At what temperature do I use the magnetic beads?

    The magnetic beads must be at room temperature before washing. After washing, the beads must be kept at room temperature for use in rRNA removal.

  • What genes are covered in the TruSight Myeloid assay?
    The TruSight Myeloid Sequencing Panel targets 54 genes (either full coding sequence or targeted exons) that are frequently mutated in myeloid malignancies. Refer to the TruSight Myeloid Sequencing Panel Data Sheet for a full list of genes.
  • What reagents are required for an instrument wash?

    The automatic post-run wash uses diluted sodium hypochlorite (NaOCl) included in the cluster cartridge. The maintenance wash, which is required every 14 days the instrument is idle, requires user-supplied dilutions of Tween 20 and NaOCl.

  • How long does it take to prepare and sequence libraries from my samples using the MiSeq FGx Forensic Genomics System?

    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.

  • What is the typical yield for this kit?

    10–200 nM/L

  • What level of sample plexity is supported for TruSeq Exome?

    The number of samples pooled pre-enrichment depends on the kit being used. See the following for sample plexity in each kit. The input amount of each library changes as plexity changes. Refer to the TruSeq Exome Library Prep Reference Guide for detailed guidance.

    Kit Name

    Catalog #

    TruSeq Exome Library Prep Kit (8 rxn x 3 plex)

    FC-150-1001

    TruSeq Exome Library Prep Kit (8 rxn x 6 plex)

    FC-150-1002

    TruSeq Exome Library Prep Kit (8 rxn x 9 plex)

    FC-150-1003

    TruSeq Exome Library Prep Kit (8 rxn x 12 plex)

    FC-150-1004

  • Are there any differences in analysis for Nextera XT samples?
    A new PCR Amplicon analysis workflow is available in MiSeq Reporter v1.3 (MSR) for the MiSeq system. The PCR Amplicon workflow requires specifying a manifest which is created in the Illumina Experiment Manager. A manifest is a list of all the targeted regions and their chromosome start and end positions. The manifest specifies regions of interest (ROIs) for the aligner and variant caller, which results in faster analysis times and visualization of results specific for only the ROIs. Note that CASAVA is not compatible with Manifest files. Please refer to the Illumina Experiment Manager User Guide (part # 15031335) for more information on sample sheet and manifest creation for Nextera XT libraries. The MiSeq software (MCS 1.2/MSR 1.3) can be downloaded from the Downloads tab of the Nextera XT Support page.
  • Why is there a higher error rate for the first few bases?
    The first two or three bases in mRNA-Seq reads have slightly elevated error rates compared to genomic DNA samples. We believe that this is an effect of the random priming process. The bases at the beginning of each read were likely at the back end of the random primer, away from the extending polymerase, during the priming process. It appears that this observation is a measurement of the mismatch pairing that is tolerated on the other end of the primer during the extension process by the polymerase.
  • Does Illumina recommend pipettes and pipette tips to use with the NeoPrep System?

    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:

    •  
      • Pipet-Lite XLS+ 8-channel LTS, 2 μl to 20 μl—Rainin, catalog # L8-20XLS+
      • One of the following 20 µl tips:
        • LTS tips 20 µl. Presterilized. Filter—Rainin, catalog # RT‑L10F
        • ART Barrier Pipette Tips 20 µl; 20 µl SoftFit-L—Fisher Scientific, catalog # 2749RI

    200 μl Volume:

    •  
      • Pipet-Lite XLS+ 8-channel LTS, 20 μl to 200 μl—Rainin, catalog # L8-200XLS+
      • One of the following 200 µl tips:
        • LTS tips 200 µl. Presterilized. Filter—Rainin, catalog # RT‑L200F
        • ART Barrier Pipette Tips 200 µl; 200 µl SoftFit-L—Fisher Scientific, catalog # 2769RI
  • What level of multiplexing is supported on the GA with Nextera? When will this be available?
    There will be 96 available barcodes using 12x8 indices, combining 12 Index 1(i7) with 8 Index 2 (i5) indices. Although kits are currently available, (the index kit supports multiplexing on all Illumina sequencing platforms) multiplexing on GA is not supported until updated SCS/RTA software is available.
  • Can I upgrade my HiSeq 2500 or earlier model to a HiSeq 4000?

    No. An upgrade package (catalog # SY-401-4002) is available for HiSeq 3000 only.

  • Do local proxies affect BaseSpace?

    No testing has been performed on the effects of local proxies on BaseSpace access.

  • What is the output file format from a sequencing run on the NextSeq system?

    The NextSeq system generates base call (BCL) files aggregated by lane with a BCL file for each lane, for each cycle. The aggregate file contains the base call and associated quality score for every cluster.

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

  • What is the recommended read length for sequencing TruSight One libraries?

    The TruSight One assay is designed to be sequenced with a 2X151 cycle paired-end sequencing run to maximize coverage of the targeted region. The design also minimizes the likelihood of sequencing into uninformative adapter sequence. TruSight One libraries use dual indices and additional 8 cycle index 1 and 8 cycle index 2 reads are necessary to demultiplex pooled enrichments. Consult the TruSight One User Guide for additional details on sample pooling and dual-indexed sequencing.

  • What has changed with the NextSeq 500/550 Kit v2?

    The NextSeq 500/550 Kit v2 provides an improved workflow during run setup. You no longer have to add sodium hypochlorite (NaOCl) and dual-index sequencing primers (BP13) manually before the run. All required reagents are included in the prefilled reagent cartridge v2.

    Reagents provided in the NextSeq 500/550 Kit v2 in combination with NCS v1.4 generate improved data quality and a higher yield of error-free reads, and enable the use of custom sequencing primers for dual-indexed runs.

  • How do I make sure that the magnetic beads are completely removed?

    Failure to remove the magnetic beads completely causes carryover of the  probes and rRNA. Place the tubes on a magnetic stand for 5 minutes, and then carefully transfer the supernatant containing the rRNA-depleted RNA to a new RNase-free tube.

  • Which export formats are available from BaseSpace Variant Interpreter?

    You can export reports in PDF file format.

  • What regions are targeted?

    The TruSeq Methyl Capture EPIC Sequencing Panel contains content that spans the full human methylome, including CpG islands, shores, shelves, enhancers, promoter regions, sites in open chromatin, gene bodies and more. This panel builds upon content included in the Infinium Human Methylation EPIC BeadChip with additional regions of importance identified by ENCODE, FANTOM5, the Epigenomics RoadMap Consortium, and customer-based requests. 

  • What IEM workflow do I use in for TruSeq Targeted RNA Expression?
    Select the RNA Resequencing category, then the Targeted RNA application.
  • How long does it take for MiSeq secondary analysis to complete?

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

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

  • What molecules are sequenced with the TruSeq Stranded mRNA Sample Prep Kit?
    All poly-adenylated molecules are isolated and sequenced. There are species of non-coding RNA that are poly-adenylated as well.
  • Are data sets available for Nextera Rapid Capture Exome Enrichment?
    Yes, there are examples of Nextera Rapid Capture Exome and Expanded Exome Enrichment data from HiSeq and MiSeq runs.
  • How long does it take to complete a 150-cycle run on the MiSeq?

    A 150-cycle run takes approximately 20 hours.

  • Is the VariantStudio software validated?

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

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

  • How will I know what are the best index combinations for my sample prep?
    The Illumina Experiment Manager (IEM) will notify you if improper combinations are used when creating a sample sheet for use with CASAVA, so it is highly recommended to create your sample sheet prior to performing sample prep/pooling. There are also low plexity pooling guidelines in the Nextera, TruSeq DNA, and TruSeq RNA sample preparation guides. Always pool samples with valid index combinations to avoid image registration failures.
  • Can I use MiSeq Reporter software to analyze data from a HiSeq system?

    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 this kit.

  • What is the difference between the tube and plate protocols? At what number of samples is it recommended to change from tube processing to plate?

    The tube protocol describes how to process samples in Eppendorf tubes and is recommended for ≤ 16 samples pre-enrichment. The plate protocol uses a 96-plate format and is recommended for when processing > 16 samples pre-enrichment.

    The tube and plate protocols require different consumables, magnetic stands, and incubation equipment. The choice of tube vs plate is user discretion and equivalent results can be expected from either option. However, the plate option can yield more consistent results between samples.

  • How long does it take to generate ready-to-sequence libraries?

    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.

  • How many boxes are included in the ForenSeq DNA Signature Prep Kit?

    The kit contains three boxes.

  • Is there sample data I can view?

    MiSeq data sets are available on the BaseSpace Public Data page.

  • Is bisulfite sequencing compatible with the TruSeq Nano DNA Library Prep Kit?

    Only LT adapters are methylated and the polymerases used in the TruSeq kits are not appropriate for bisulfite applications.

  • Is this kit single index or dual index?

    This kit is single index.

  • How does BaseSpace Variant Interpreter handle tri-allelic sites?

    When both alleles of a heterozygous position are different from the reference, as in a tri-allelic position, the variants are split into 2 lines and both variants are annotated.

  • How are base calling and quality scoring performed on the NextSeq system?

    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:

    • Template generation—Defines the position of each cluster on the flow cell.
    • Imaging—In real time, images are passed in memory to RTA v2.
    • Base calling—Determines a base for every cluster at a specific cycle. After a base is called, RTA v2 assigns a quality score. The result is written to base call (BCL) files, which are aggregated in one file per lane per cycle.
  • Why do I need to use a keyed 8-tube strip for libraries?

    Using the keyed 8-tube strip is important for sample tracking. Use Seahorse Bioscience catalog # 204026-100, which provides keyed 8-tube strips with barcode labels designed specifically for use on the cBot 2.

  • What is the recommended DNA concentration for cluster generation?

    The first time you process template DNA, try a concentration titration range to optimize the number of clusters formed. If the concentration is too low, fewer clusters are generated and result in a low sequencing yield. If the concentration is too high, clusters are too dense and can complicate data analysis. For more information, see the Sequencing Library qPCR Quantification Guide (part # 11322363) (a MyIllumina login is required) or the HiSeq and GAIIx Systems Denature and Dilute Libraries Guide (part # 15050107).

  • Can variant classification information be versioned in the Classification Database?

    No, variant classification and associated notes saved in the Classification Database are not versioned. If the classification category or notes are modified for a given variant in the Classification Database, previous information is overwritten with the current modification.

  • Will I have enough reagents in my 200-Cycle SBS Kit to perform a 2x100 cycle run with indexing?
    Yes. The TruSeq SBS Kit (200 Cycles) contains sufficient reagents for 209 cycles of sequencing, which covers 101 cycles for Read 1, 7 cycles for the Index Read, and 101 cycles for Read 2.
  • What sequencing platforms and reagents is compatible with this kit?

    This kit is compatible with the MiSeq, NextSeq, and HiSeq platforms, excluding the HiSeq X platforms. Plexity recommendations are based on the kit size: 24 sample kit = 8 enrichments x 3 libraries per enrichment, 48 = 8 x 6, 72 = 8 x 9, 96 = 8 x 12 .

  • Can I use the Nextera XT kit on all Illumina sequencing platforms?
    Although the Nextera XT kit has been optimized for use on the MiSeq system, this kit can be used on any Illumina sequencing platform. However, please note that CASAVA is not compatible with Manifest files and therefore is unable to analyze data generated on the HiSeq, HiScanSQ or GAIIx platforms.
  • What DNA input amount is required for the TruSight DNA Amplicon assay?
    The assay uses 50 ng of intact, high-quality genomic DNA. DNA extracted from Formalin-Fixed, Paraffin-Embedded (FFPE) tissue is not supported.
  • Can I perform family-based genetic disease analysis in BaseSpace Variant Interpreter?

    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.

  • How long does it take to complete a water wash and maintenance wash?

    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.

  • Exactly which files are uploaded as instrument health data?

    The files uploaded as instrument health data are RunInfo.xml, RunParameters.xml, RTAComplete.txt, InterOp files, and RTAConfiguration.xml.

  • Which reagent position is used for the MiSeq flow check?
    During the pre-run flow check, reagent is pulled from position 3 on the reagent cartridge, which is PR2.
  • Can GenomeStudio software display non-human sequence data?
    Yes. GenomeStudio can display data from non-human genomes.
  • How do I send run data to BaseSpace?

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

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

  • What is Adapter Trimming?
    Shorter inserts may lead to sequencing into the adapter, and this feature helps filter out adapter sequence from the final sequence data. Please select this option when creating your sample sheet in IEM for use with MiSeq as the MiSeq Reporter (MSR) analysis software will automatically trim the adapter sequence. For all other run types, this option can be used with the appropriate commands in CASAVA 1.8.2.
  • If I choose to design my project with SNP avoidance turned off, what effect will it have on my data?

    If an upstream or downstream amplicon primer region overlaps an actual variant in the input genomic DNA, the assay can experience reduced specificity and/or uniformity relative to other amplicons in the multiplexed reaction.

  • How many additional cycles of SBS reagents do I need to calculate into the run for sequencing dual-indexed libraries?

    For dual index paired-end runs, there are 23 additional cycles (index & chemistry only).
    For dual-index single-read runs, there are 16 additional cycles of indexing.
    For information about the number of SBS kits required on the HiSeq, HiScanSQ, or GAIIx, see the user guide for your instrument guide.

  • What is a TruSeq Targeted RNA Expression fixed content project?
    Fixed content projects are pre-synthesized oligo pools that you can order. These have been wet-bench tested and designed against various cellular and disease-causing pathways.
  • Why is directional sequence information useful?

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

  • Does the HiSeq Analysis Software replace CASAVA?

    No, the HiSeq Analysis Software provides analysis of libraries prepared with the Nextera Rapid Capture exome enrichment kit and human Whole Genome Sequencing using only the hg19 genome as a reference. CASAVA provides analysis of additional applications such as RNA sequencing, Exome sequencing, targeted resequencing, and Whole Genome Sequencing using a more extended set of reference genomes available on Illumina.com's iGenomes page. (http://support.illumina.com/sequencing/sequencing_software/igenome.ilmn). More information on CASAVA is available here: http://www.illumina.com/software/genome_analyzer_software.ilmn#casava

  • Is VariantStudio available as a command-line tool?

    No. Illumina VariantStudio was is a point-and-click software application that enables variant data exploration, annotation, and filtering without requiring bioinformatics expertise.

  • Can TruSeq adapters be used for methyl-Seq?

    TruSeq adapters are methylated.  However, the kits are not currently compatible for the following reasons:

    • WGBS:  The PMM (polymerase mix) used in the TruSeq DNA Sample Prep kits cannot handle uracil bases in its template.  This means that bisulfite-converted DNA cannot be processed directly with TruSeq kits, unless another polymerase is used to amplify the samples.
    • RRBS: Same as above, and in addition the length of TruSeq adaptors results in the generation of adaptor dimers that overlap the size range of the targeted DNA, making them incompatible with this application.        
  • Do homopolymers and repetitive DNA regions impact sequencing efficiency?
    Homopolymers do not impact sequencing. The number of uniquely alignable reads is a function of the repeat content, so this will have an impact on productivity. With longer reads and paired-end sequencing, this may be less of an issue.
  • Can custom primers be used on the HiSeq 4000?

    Custom primers have not been tested for use on the HiSeq 4000.

  • What data quality can I expect from a run on the HiSeq 4000?

    You can expect greater than 75% of all bases above Q30 with a 2 x 150 bp run. For more information, see HiSeq 3000/HiSeq 4000 System Specifications.

  • Does Illumina have any recommendation about ChIP methods for TruSeq ChIP sample prep?

    Illumina does not provide any recommendations as methods for ChIP pulldown and fragmentation are dependent upon individual antibodies and procedures. Please reference literature or other sources for recommendations.

  • What is instrument health data, and where can I find more information about the option to send instrument health data to Illumina?
    HCS v2.2 allows HiSeq instruments connected to the internet to send instrument health information to Illumina. This information is anonymous and includes only generic run metrics. This information is used by Illumina to help improve Illumina products. If you want to turn off this option or would like further information, see the Options menu in the HiSeq Control Software. You can find the Options menu under Menu, then Tools.
  • Are AMPure XP beads supplied with TruSeq Library Prep kits?

    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.

  • What do I need to know to allow VariantStudio access to BaseSpace via my company firewall?

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

  • How many tiles are imaged on a HiSeq 3000/4000 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.

  • How do I determine which version of GenomeStudio software I am using?

    With GenomeStudio software open, go to the Help menu and select About. The About screen includes GenomeStudio software version information.

  • How do I know if I should be concerned about the Nextera Rapid Capture Custom Enrichment design in my targeted regions?
    DesignStudio identifies Design Warnings to alert you to designs that are targeting areas of the genome that carry a higher performance risk. These warnings (e.g., low specificity, poor GC content) are displayed in the Regions, Targets, and Probes tables in DesignStudio.
  • Can I add genes or targets of interest to the TruSeq Amplicon - Cancer Panel?

    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.

  • Where can I find information about library input for the various HiSeq chemistries?

    See Denaturing and Diluting Libraries for the HiSeq and GAIIx (part # 15050107).

  • Can I perform Single Read runs and still get both TruSeq HT library prep index sequences?

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

  • How many samples can I process at a time?

    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.

  • What type of library prep is supported on the HiSeq 4000?

    Like the HiSeq 2500, the HiSeq 4000 is designed as an open platform intended to support a broad array of applications. Internally, Illumina has run numerous library prep kits without issue. For sample data demonstrating system performance, see the HiSeq 3000/4000 Sample Datasheets.

    The HiSeq 4000 system is compatible with current paired-end P5 and P7 primers. Single-read and paired-end libraries are compatible with the HiSeq 4000, but must use current adapters. Legacy libraries that are based on the original Illumina single-read adapter do not cluster properly.

  • Can I use HiSeq or Genome Analyzer reagents on the MiSeq?

    No. MiSeq kits use an all-inclusive reagent cartridge system that is specifically designed to deliver the reagents necessary for cluster generation, sequencing, and paired-end chemistry.

  • Does cDNA cluster in the same way as gDNA?

    cDNA libraries should be considered an equivalent to gDNA for clustering and sequencing steps, assuming an equivalent complexity of the sample.

  • Can CASAVA be used to analyze MiSeq data offline?
    Yes, CASAVA can be used to analyze MiSeq data offline.  The MiSeq output folders are readable by CASAVA without a need to modify any configuration files.
  • Do I have to use an x-tracta tool for the excision of the gel band during TruSeq Synthetic Long-Read DNA library prep?
    The x-tracta has been validated by Illumina, however any gel extraction tool or band excision will work.
  • Why is bisulfite conversion performed after adapter ligation instead of before adapter ligation?

    Illumina chooses to pull down target regions before bisulfite conversion so that probes can be designed to specifically bind to their target regions. After bisulfite conversion, unknown sequences are abundant because every cytosine could be either a cytosine or a thymine (or uracil depending on if PCR occurs before target capture).  Designing probes to unknown sequences would incorporate some bias for either methylated or unmethylated sequences. To avoid possible bias during target capture, we perform target pulldown before bisulfite conversion.

  • Does Isaac utilize quality information during SNP-calling?
    Base qualities are considered during mapping to calculate the alignment score and during SNP and indel calling to calculate the variant quality scores.
  • Is it possible to generate a larger insert size with TruSeq RNA Sample Prep?
    See the appropriate TruSeq RNA sample preparation guide for details, Appendix A, Alternate Fragmentation Protocols. Two separate options are provided for varying the insert size of your library:

     

    1. Modify the fragmentation time
    2. Shear the sample after the synthesis of the double-stranded cDNA
  • What are the effects of putting too much or too little genomic DNA into the TruSeq Rapid Exome protocol?

    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.

  • What is the difference between TruSight Tumor 15 and TruSight Tumor 26?

    TruSight Tumor 15 targets 15 solid tumor genes, while TruSight Tumor 26 targets 26 genes. TruSight Tumor 15 is a multiplex PCR amplicon based assay and the TruSight Tumor 26 utilizes the TruSeq Custom Amplicon extension-ligation chemistry. 

  • What software is needed to run TruSeq Synthetic Long Read libraries?
    • IEM 1.8 or later is required to create a sample sheet.
    • cBot 1.4 software or later (v3) or cBot 2.0.1 or later (v4) may be needed for cluster generation.
    • HCS 2.0/RTA 1.17 or later is required for sequencing.
    • The BaseSpace TruSeq Long-Read Assembly App or TruSeq Phasing Analysis App are required for analysis.
  • Will the TruSeq Stranded mRNA Sample Prep Kit work for prokaryotes?

    The mRNA isolation protocol relies on pulling out RNA species that have a poly-A tail. Since prokaryotic mRNA is not polyadenylated, Illumina's TruSeq Stranded mRNA Sample Prep kits are not suited for these organisms. Please see Epicentre's portfolio of Ribo-Zero products for kits optimized for prokaryotic organisms.

  • Which species are compatible?

    The Ribo-Zero kits were developed using model organisms. Results can vary depending on the species from which the RNA sample was derived, especially with 5S rRNA removal.

    Visit the RNAMatchMaker tool, www.epibio.com/rnamatchmaker, on the Epicentre website to confirm species compatibly.

  • Can I pool data from different TruSeq Targeted RNA Expression runs together if they used the same TOP pool?
    No. Refer to the TruSeq Targeted RNA Expression tech note for information to determine the appropriate number of samples to be sequenced on a flow cell. For assays with low counts (< 10), pool fewer samples so that the read budget is increased for the low count samples.
  • What happens if my data connection is interrupted during a run on the NextSeq system?

    In the event that data transfer is interrupted during a run, data are stored temporarily on the instrument computer until the connection is restored. When the connection is restored, transferring of data resumes automatically.

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

  • Can I freeze/thaw a TruSeq HT sample prep adapter plate?

    Yes, the adapter plate can undergo freeze/thawing up to 4 times. More freeze thaws of the plates may impair assay performance.

  • How will I know what are the best index combinations?
    The Illumina Experiment Manager (IEM) will notify you if improper combinations are used when creating a sample sheet, so it is highly recommended that you create your sample sheet prior to performing sample prep/pooling. There are also low plexity pooling guidelines in the Nextera DNA Sample Preparation User Guide. Always pool samples with valid index combination to avoid image registration failures.
  • How does VariantStudio handle tri-allelic sites?

    When both alleles of a heterozygous position are different from the reference, as in a tri-allelic position, the variants are split into two lines and both variants are annotated.

  • Do I need oil to load a flow cell on the HiSeq or HiScanSQ?
    No. The HiSeq and HiScanSQ do not require immersion oil to properly load a flow cell in the way that the Genome Analyzer does. Instead, the flow cell is held in place by a vacuum, which removes air and replaces the need for immersion oil.
  • Does Illumina offer a custom targeted resequencing product?

    Yes. Illumina offers a TruSeq Custom Enrichment kit. Reference the TruSeq Custom Enrichment Data Sheet for more information.

  • Why is it recommended to run 2 x 101 cycles for Rapid Run but only 2 x 100 cycles for High Output runs for TruSeq Synthetic Long-Read DNA library sequencing?
    SBS v3 kits support 209 cycles. Sequencing 2 x 101 cycles + 8 bp index cycles cause the R2 to run out of reagents, unless additional SBS kits are stacked for >209 cycles.
  • What if I did not name the sample sheet with the reagent cartridge barcode number?
    During run setup, the software searches for a sample sheet of a name that matches the barcode number of the reagent cartridge scanned in the previous run setup step. If a sample sheet of this name is not found, the software will pause and prompt you to browse to the apppropriate sample sheet for this run, regardless of the sample sheet file name. Naming the sample sheet with the reagent barcode number skips this additional step.
  • What is the HiSeq Analysis Software?

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

  • Are there any protocol considerations for the TruSeq Stranded Total RNA Sample Prep Kits with Ribo-Zero Globin or Plant?
    All Ribo-Zero kits follow the same protocol in the TruSeq Stranded Total RNA Sample Preparation Guide. The only component that differs in each kit variation is the rRNA Removal Mix.
  • Will cBot plates be updated with the sequencing primers required for dual-indexed Nextera libraries and TruSeq HT libraries?

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

  • I can’t see a reference sequence in the IGV (Illumina Genome Viewer) or ICB (Illumina Chromosome Browser). How can I display a reference sequence?

    See Chapter 5 of the GenomeStudio 2008.1 Framework User Guide, available on iCom and in the GenomeStudio Portal.

  • How do I manage DNA contamination?

    RNA samples must be trated with DNase before starting the rRNA removal protocol. Otherwise, the probes can bind to the DNA and reduce rRNA removal.

  • Can my sample report templates be used in other VariantStudio projects?

    Yes. Report templates created and saved in VariantStudio are available to other VariantStudio projects.

  • I got a warning message in HCS about the Tdi Scan. What does this mean and what should I do?

    TDI Scan warning messages indicate an issue with image acquisition and storage; however, the system will automatically retry image capture to self-correct. TdiScan messages usually have no effect on the run other than slightly extended cycle times, and do not affect the run data as images are re-captured before continuing.

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

  • Does BaseSpace require a sample sheet?

    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.

  • What are the safe stopping points in this protocol?

    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.

  • Why is my SBS v3 reagent waste brown?

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

  • How long can HiSeq v4 reagents be stored after initial preparation?

    Prepare HiSeq v4 SBS reagents the night before or on the same day of use only. Do not store reagents longer than overnight. For use on the same day, store prepared reagents on ice in the original bottle with the cap tightened. For use the next day, store prepared reagents at 2°C to 8°C overnight.

  • If a network outage to the central file server storing a runs occurs, can NovaSeq cache the entire 2 x 150 run?

    Yes. The server can store as many runs as disk space permits. Real-Time Analysis continues processing and resumes data transfer when the network is restored.

  • How much DNA is required to load a flow cell lane for bridge PCR?
    If possible, start the sample prep with 1–5 μg of DNA, although 1–2 μg is enough for many flow cells.
  • What is the difference between the TruSeq Methyl Capture EPIC LT and HT kits?

    TruSeq Methyl Capture EPIC LT and HT kits are kitted for different sample numbers.  

    Kit

    Sample Number

    Enrichment
    Reactions

    Indexes

    TruSeq Methyl Capture EPIC - LT

    12

    3

    4

    TruSeq Methyl Capture EPIC - HT

    48

    12

    12

  • What are the lab temperature requirements for the HiSeq?
    The lab should maintain a temperature of 19-25°C (22°C ±3°C). This is the operating temperature of the instrument. During a run, do not allow the ambient temperature to vary by more than ±2°C. Maintain a relative non-condensing humidity between 20-80%.
  • What is the difference between the TruSight One and Nextera Rapid Capture (NRC) Exome and NRC Expanded Exome products?

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

  • Can Sequencing Analysis Viewer (SAV) be used with the MiSeq to view primary analysis results?
    Yes. You can install SAV on another computer that is connected to the same network as the instrument. MiSeq data will appear when you select All or Lane 1 from the drop-down menu. For more information, see the Sequencing Analysis Viewer User Guide.
  • Which metrics can be used to evaluate data in the SNP Table to identify SNPs that require further editing and poorly performing SNPs?

    SNPs can be evaluated and sorted by Call Freq, # no calls, Poly 10%, and Poly 50%.

  • How much time does a standard HiSeq run take?

    See system specifications on the HiSeq 2500 Specifications page.

  • If I run two flow cells at the same time, do they need to be identical in setup?

    No. You can start each flow cell independently from the other. Each flow cell can have a different number of reads and cycles.

  • What is the difference between Nextera Enrichment and Nextera Rapid Capture Enrichment?

    Nextera Rapid Capture Enrichment is an updated, faster enrichment workflow that offers a more efficient exome (45 Mb) in addition to the more comprehensive existing exome product (62 Mb).

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

  • Does VariantStudio run on a Mac or Linux system?

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

  • What operating system is used on the MiSeq?

    The MiSeq runs on Windows 7 Embedded Standard.

  • What power connections are required for the NextSeq system?

    The NextSeq instrument comes with a region-specific power cord. For more information, see the NextSeq System Site Prep Guide.

  • What is the difference between TruSeq Exome and TruSeq Rapid Exome?

    TruSeq Rapid Exome uses transposon-based fragmentation while TruSeq Exome uses an alternate enrichment protocol that uses mechanical shearing. 

    The TruSeq Exome method makes it suitable for DNA that does not respond well to Nextera tagmentation. The TruSeq Exome protocol is a combination of the proven TruSeq Nano Library Prep and Rapid Capture Enrichment kits. 

  • Has the tile layout or numbering changed due the wider lanes on Flow Cell v3?

    No, the tile numbering is unchanged from the format introduced in HCS v1.3. However, when using Flow Cell v3, the tile numbering reflects the three-swath imaging pattern, where a 3 in the tile number represents the third swath.

  • What is the alternative SBS workflow?

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

  • What DNA input amount is required?

    The assay uses 50ng of DNA.

  • What do I do if my AMPure beads are not dry after the 15 minute incubation at room temp?

    Longer drying times may be required depending on environmental variables and the amount of ethanol remaining in the well.  However, take care not to over-dry beads as this can impact sample recovery.

  • Do I need to purchase the dual index primer reagent box for sequencing these libraries?

    The TruSeq Dual Index Sequencing Primer Kit (catalog #PE-121-1003) is required for clustering libraries on the cBot and paired-end sequencing on the HiSeq 2000, GAIIx, and in High Output mode on the HiSeq 2500. This kit is good for one run and contains the required primers for dual 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.

  • Can larger DNA inserts be selected during the gel size selection step of the TruSeq ChIP Sample Prep protocol?

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

  • Is a wash required before or after scanning a BeadChip?

    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.

  • Can the wells of a TruSeq HT library prep adapter plate be reused?

    No, the wells cannot be reused.

  • How are the TruSight RNA Pan-Cancer and TruSight RNA Fusion components different?

    Boxes 1-4 are identical.

    Box 5 of the TruSight RNA Pan-Cancer Panel kit contains an oligo tube targeting 1385 genes.

    Box 5 of the TruSight RNA Fusion Panel kit contains an oligo tube targeting 507 genes.

  • What is an amplicon and what is the size of an amplicon?

    An amplicon is a fragment of DNA that is produced from an amplification event such as PCR. Amplicon sizes are user-selected when creating a project in DesignStudio.

    Amplicon sizes can be 150, 175, 250, or 425 bp in size.

  • What is bcl2fastq?

    The bcl2fastq v1.8.4 conversion software is a separate piece of standalone software that is run on a Linux scientific computing system. The installer can be downloaded from the Illumina website. System requirements are outlined in the bcl2fastq User Guide (part # 15038058). If BCL files are zipped, then the use of the bcl2fastq v1.8.4 is required.

  • Is it possible to scan a BeadChip on the NextSeq 550 system when a wash is due?

    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.

  • How do I confirm that a fusion is real?

    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.

  • Are there restrictions to the Minimum Number of Points in Cluster?
    This number can be any number different from 0, however we recommend that the Minimum Number of Points in Cluster is set to match the biology of your samples and size of your dataset.  A general guideline is to set this number to 1-4% of the number of samples that are performing well in the data set.
  • Can GenomeStudio be used to view MiSeq data?

    No. MiSeq Reporter and the Illumina Amplicon Viewer are used to view MiSeq data. For an overview of the software, see the MiSeq System software page.

  • What Illumina sequencing platforms are compatible with TruSeq ChIP libraries?

    TruSeq ChIP libraries are compatible with all Illumina sequencing platforms, including MiSeq, HiSeq, HiScanSQ and Genome Analyzer.

  • Which Illumina sequencing platforms are compatible TruSeq RNA Access libraries?
    TruSeq RNA Access Library Prep libraries are supported on all Illumina sequencing platforms.
  • What are the effects of putting too much or too little genomic DNA into the TruSight One protocol?

    Overloading the Nextera tagmentation reaction with more than 50 ng of genomic DNA leads to a larger library size distribution, which can lower the number of on-target sequencing reads. Less than 50 ng of input DNA leads to a smaller library size distribution. The result is reduced library diversity or elevated duplicate reads  because many of these smaller library sized fragments would be eliminated during the size-selection procedures.

  • Can I use Illumina VariantStudio with data from other sequencing platforms?

    No. The Illumina VariantStudio End User License Agreement (EULA) states that Illumina VariantStudio must be used solely to analyze data generated from an Illumina sequencing instrument.

  • Where can I find the manifest file for the TruSight One Sequencing Panel Kit?

    The TruSight One manifest can be downloaded from the Downloads tab of the TruSight One support page on support.illumina.com.

  • Which reagents or kit components should not be freeze thawed?

    All reagents that come frozen can withstand the freeze thaw process.  Components such as the magnetic beads and SPM should not be frozen at any point.

  • Are flow cells provided in MiSeq v3 kits different from flow cells provided in MiSeq v2 kits?
    No. The flow cells are identical. However, the upgrade to MCS v2.3 enables imagine of the flow cell in 19 tiles per surface. MCS v2.3 is required software for the MiSeq Reagent Kit v3.
  • How much input genomic DNA is required? Can I use more or less than the recommended amount?

    500 ng of input genomic DNA is required.

    Illumina has not validated the workflow with less than 500 ng.  Illumina has observed that for less than 500 ng of input DNA, a corresponding increase in duplicates were observed. Therefore, libraries may require increased sequencing depth to attain the desired level of coverage.

    Illumina has tested up to 1 µg which has produced fewer duplicates and therefore better coverage.

  • What do I need to order if I want to run a new Illumina Nextera library?
    • The Illumina Nextera DNA Sample Preparation Kit comes in two sizes: a 24 Sample Kit (Catalog # FC-121-1030) and a 96 Sample Kit (Catalog # FC-121-1031).
    • To complete the sample prep protocol, there are two Index Kits used for PCR: a 24 Index Kit with sufficient reagents for 96 samples (Catalog # FC-121-1011) and a 96 Index Kit with sufficient reagents for 384 samples (Catalog # FC-121-1012). Again, note that each Index Kit is good for four uses and additional caps are included for the tubes to replace after each use in order to prevent cross-contamination.  To assist in correctly arranging index primers during the PCR Amplification steps, a TruSeq Index Plate Fixture Kit is available to order (Catalog # FC-130-1005).
    • When clustering Illumina Nextera libraries on the Cluster Station or cBot and sequencing on a HiSeq 2000/1000, HiScanSQ or GAIIx, new primers are required whether performing a non-indexed, single-indexed, or dual-indexed run. There are two accessory kits available for this: the TruSeq Dual Index Sequencing Primer Kit for Single Read runs (Catalog # FC-121-1003) and the TruSeq Dual Index Sequencing Primer Kit for Paired End runs (Catalog # PE-121-1003). Note that each kit is good for a single run but contains the required primers for both clustering and sequencing.
    • When preparing Illumina Nextera libraries for clustering and sequencing on MiSeq, the appropriate primers are already included in the reagent cartridges: MiSeq 50-cycle Reagent Kit (Catalog # MS-102-1002) or MiSeq 300-cycle Reagent Kit (Catalog # MS-102-1001).
  • Why are spare caps provided with the index tubes in the ForenSeq DNA Signature Prep Kit?

    Caps are provided to prevent cross-index contamination.  After a cap has been removed from a tube, it is discarded and a fresh cap is used to seal the tube after use.

  • What is the shelf life of the TruSeq HT sample prep kits?

    One year from the date of manufacture. The kit will contain an expiration date on the label.  Illumina guarantees at least 3 months from the date of receipt. This is the same as the TruSeq DNA LT and TruSeq RNA v2 kits. 

  • Why do I have to log into my BaseSpace account to annotate variants in VariantStudio?
    Illumina uses BaseSpace authentication to make sure that you are authorized to use the application. After you have authenticated for the first time, VariantStudio saves your credentials so that you are not asked to log in again later. You can delete your credentials using Annotation & Classification | Annotation Options | Forget BaseSpace Logon.
  • Why are the TruSeq Stranded Total RNA Sample Prep Ribo-Zero instructions different for Illumina protocols compared to Epicentre’s protocol?

    Illumina has optimized the workflow to increase ease-of-use and scalability. Changes include the use of plates compatible with multi-channel pipettes, the streamlining of incubation steps, and an overall reduction in hands-on time.

  • How long does the cBot take to perform clustering on a HiSeq v4 flow cell?
    Clustering takes slightly more than 2 hours. Clustering on a HiSeq v4 requires the updated cBot software (v2.0.16, or later) and requires v9.0 recipes.
  • Can rapid run SBS reagents and high output SBS reagents be combined?

    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.

  • What are TruSight DNA Amplicon libraries?
    TruSight DNA Amplicon libraries are fixed content panels that leverage Illumina’s TruSeq Custom Amplicon (TSCA) assay. TSCA is the fastest and easiest multiplexed amplicon assay optimized for the MiSeq System. It allows for the sequencing of 16–1536 custom amplicons (small regions of interest) across up to 96 samples. The assay also includes on-instrument variant calling for somatic variation with the Somatic Variant Caller on the MiSeq System.
  • What files do I need to scan a BeadChip on the NextSeq 550?

    To perform a scan on the NextSeq 550, you need Decode (*.dmap) files, a manifest  (*.bpm) file, and a cluster (*.egt) file for the BeadChip you are using. The Decode files are unique for each BeadChip barcode. The manifest and cluster files are unique to the BeadChip product type.

  • What is an oxo Q-score?

    During enrichment, C to A mutations in the DNA can occur. The oxo Q-score is a way of measuring the rate of these mutations.  This protocol has been optimized to minimalize these mutations by using a proprietary buffer.

  • How often do I need to perform an instrument wash on the NextSeq 550 system?

    The NextSeq software performs an automatic post-run wash on the NextSeq 550 system after a successful sequencing run.

    • If the reagent cartridge and buffer cartridge are in place and the instrument is idle, a quick wash is required every 14 days.
    • If the instrument is in a dry state, for example only performing array scans using the BeadChips, and the reagent cartridge and buffer cartridge are removed, a quick wash is required every 7 days. 
  • What is the recommended run length and type for TruSeq stranded RNA sample prep libraries?

    The read length and format (single read versus paired end) are important considerations in the design of RNA sequencing experiments. The table below provide guidance on some factors to consider. These recommendations are based on internal and external data, but do not represent strict cut-offs. Needs for individual projects may vary based on multiple variables as well as user preference.

    Applications

    Read Type (bp)

     Read Depth/Sample (mRNA/Total RNA)

    Gene profiling (gene-level counts)

    1 x 50

    >5 m / >10 m

    Discovery (alternative transcripts, gene fusions, etc.)

    2 x 50 - 75

    ≥50 m / >100 m

    Complete transcriptome annotation

    2 x 75 - 100

    ≥100 m / ≥200 m

  • How is scanning enabled on the NextSeq 550 system?

    Array scanning is enabled out of the box on the NextSeq 550 system.

  • How long can NeoPrep libraries remain on the instrument?

    Libraries can remain at room temperature on a library card for up to 3 days after a run is complete.

  • What are Illumina's recommendations for preparation of ChIP DNA?

    We do not have any specific recommendations regarding the chromatin immunoprecipitation step, as techniques for this process can vary widely based on the desired application. However, we do not recommend using any type of nucleic acid as a carrier in the chromatin immunoprecipitation prior to sequencing. The carrier nucleic acid is difficult to remove from the sample, and can end up being 50–90% of the final library.

  • How much input genomic DNA is required for the Nextera rapid Capture Enrichment workflow?
    50 ng of input genomic DNA is required. It is important to accurately quantify the input genomic DNA to generate a high quality library of the correct size.  Illumina highly recommends using a fluorometric-based quantification method for the input genomic DNA. For more information, see the DNA Input Recommendations section of the Nextera Rapid Capture Enrichment Guide.
  • Is the version of MiSeq Reporter software used for analysis recorded in the run folder?
    The MiSeq Reporter software version can be found in the following files located at the root level of the run folder: the log file AnalysisLog.txt, the CompletedJobInfo.xml file, and the workflow-specific results file (e.g. ResequencingRunStatistics.xml).
  • What method is required to quantify my input genomic DNA for ForenSeq DNA Signature Prep?

    A fluorometric based method, such as qPCR is recommended for quantifying gDNA.

  • Is quantitation performed before or after pooling TruSeq Targeted RNA Expression libraries?
    Samples are quantified after pooling. For more information, see the TruSeq Targeted RNA Expression Guide.
  • Can I use the same reagents on the HiSeq that I use on my Genome Analyzer?
    No. Cluster Kits and SBS Kits for the HiSeq are not equivalent or compatible with the Genome Analyzer. Likewise, Cluster Kits and SBS Kits for the Genome Analyzer are not equivalent or compatible with the HiSeq.
  • What is the read length on MiSeq?

    You can perform up to 2 x 250 bp, or 500 cycles of sequencing on the MiSeq using the MiSeq Reagent Kit v2. However, as few as 36 cycles can be used for some applications.

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

  • What does a paired read and a split read mean in the Fusion Calls table from the BaseSpace RNA-Alignment App results?

    A paired read is a fusion where 1 read aligns to the left gene and the other read aligns to the right gene.

    A split read is a fusion where 1 of the reads spans the fusion junction.

  • How many transcripts can I target with TruSeq Targeted RNA Expression?
    Your order can contain 12-1000 probes.
  • How many cycles can I perform with the MiSeq Reagent Kit?
    Illumina offers a variety of kit sizes ranging from a 50-cycle kit to a 600-cycle kit. MiSeq Reagent Kit v3 is available in sizes of 150 cycles and 600 cycles. The MiSeq Reagent Kit v2 is available in sizes of 50 cycles, 300 cycles, and 500 cycles. For more information, see the MiSeq Reagent Kit support page.
  • I am currently using the TruSeq DNA Sample Prep Kit. Should I switch to the TruSeq DNA PCR-Free Library Prep Kit or the TruSeq Nano DNA Library Prep Kit?

    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 TruSeq DNA PCR-Free Library Prep Kit requires 1 µg of gDNA, while the TruSeq Nano DNA Library Prep Kit requires 100 ng of gDNA.
    • The TruSeq DNA PCR-Free kit delivers the utmost data quality by eliminating PCR-induced biases, specifically, greater coverage of hard to reach regions, such as fosmid "difficult" promoters.
    • The TruSeq Nano DNA kit generates premier data quality, superior to TruSeq DNA, however it does have some PCR-induced bias and PCR duplicates, and cannot cover "difficult" promoters as well as TruSeq DNA PCR-Free.
    • TruSeq Nano DNA final libraries can be quantified using either qPCR or a fluorometric method using dsDNA binding dyes such as Qubit or Picogreen, while TruSeq DNA PCR-Free final libraries can only be quantified using qPCR.
  • When should you use TruSight RNA Pan-Cancer and when should you use TruSight RNA Fusion?

    The TruSight Pan-Cancer panel is a 1385 gene RNA panel targeting customers who want a comprehensive picture of the changes occurring in cancer, with the ability to detect gene fusions.

    TruSight RNA Fusion covers most cancer-associated fusions genes (507 genes) and enables customers to process all samples with the same workflow, run 8 samples per run on a desktop sequencer, and obtain a simple report of identified fusions at an economical price point. 

  • What is the amplicon size in the TruSight Myeloid assay?
    TruSight Myeloid amplicons are 225–275 bp in length.
  • Can Illumina Nextera DNA Sample Preparation kits be used for methylation studies?
    No, because the adaptors are not methylated.
  • Do I need to begin clustering with one of the two default algorithms, OPTICS or DBSCAN?
    No, you can also cluster by defining #clusters, if known based on the biology of your samples.
  • I cannot find a Report Wizard in the PC Module. How can I create reports from my data?

    Data can be exported directly from the Samples Table, SNP Table, and Full Data Table for downstream analysis. Mark the columns and rows you wish to export and click the icon for "Export displayed data to file" to save selected table content in *.txt or *.csv format.

  • Is there a 300-cycle MiSeq v3 reagent kit?

    No. Illumina recommends purchasing a 600-cycle kit and discarding the excess reagents after the run. Because of the increased cluster density enabled by v3 runs, this is more economical than running fewer samples with MiSeq v2 reagents.

  • Do I need to normalize my data for TruSeq Targeted RNA Expression?
    Yes, you will need to select internal normalizer genes from RNA-Seq or Microarray data from samples similar to those that will be run on the assay. The genes should be invariant across the samples being tested. Assays specific to the normalizer genes need to be included in the final TOP panel.
  • What is the reference database for ADT: i.e., the source and version for sequence, Minor Allele Frequency (MAF), and validation?
    ADT retrieves data based on dbSNP126 for sequence, position, and MAF.
  • Do I need to purchase Nextera sequencing primers separately?
    The correct primers are already included with MiSeq cartridges. However, the Nextera sequencing primers will need to be purchased separately when sequencing libraries on HiSeq2000, HiSeq 1000, HiScanSQ, or GAIIx. These primers are included in the TruSeq Dual Index Sequencing Primer Kits: the Single Read Kit is Catalog # FC-121-1003 and the Paired End Kit is Catalog # PE-121-1003.
  • Does the TruSeq Stranded Total RNA Sample Prep Kit with Ribo-Zero Plant kit work on plant leaves only, or is it also compatible with seeds and roots?
    The Ribo-Zero Plant kit contains oligos that will remove cytoplasmic and chloroplast rRNA from leaves, seeds, and roots.
  • Are the RFID tags for the flow cell and reagent cartridge linked in any way?
    No. The flow cell RFID is unique to the flow cell and reagent cartridge RFID is unique to the reagent cartridge.
  • Is the VariantStudio software a clinical software tool?

    No. The VariantStudio software was developed as a Research Use Only tool.

  • Are somatic variants being called by the Isaac Variant Caller?
    Only diploid variant calls are detected.
  • Can I use a TruSeq DNA sample prep kit with a TruSeq DNA PCR-Free library prep protocol? Can I use a TruSeq DNA PCR-Free Library Prep Kit with a TruSeq DNA sample prep protocol?

    No, kits are not interchangeable.

  • When changing parameters in the Clustering Options dialog box, will this impact or change the existing clustered data?
    No, the new settings will only be applied to any SNPs clustered after applying changes.
  • Why can't I find my ChIP-Seq data when I start a ChIP-Seq project in GenomeStudio?

    The project creation wizard initially looks in the data repository directory, not the directories containing individual runs or projects, so you must point it at the correct directory. If there are sorted.txt files in multiple locations, such as \chip-data\run1\GERALD\sorted.txt and \chip-data\run2\GERALD\sorted.txt, then you should direct the wizard to \chip-data. It will search through any subfolders and display available runs.

  • What is the minimum number of samples that I can multiplex with my Nextera Rapid Capture Custom Enrichment probe pools?
    Post-enrichment library yields may be insufficient for clustering and sequencing if doing low-levels of multiplexing of small capture target panels. Illumina recommends avoiding low levels of sample multiplexing (e.g., < 6-plex) for a small capture target size (e.g., < 2 Mb). For large custom designs multiplexing down to 1x can be achieved.
  • How much starting material does the ChIP-Seq Sample Preparation Kit require?

    The ChIP-Seq Sample Preparation Kit is designed to use 10 ng of ChIP-enriched DNA. Many users have reported that using at least 30 ng of DNA makes the protocol simpler and more repeatable.

  • Can Nextera handle sample indexing, and how many indices does Nextera support?
    If you would like to index Nextera prepared samples, there are two Nextera Index Kits: a 24 Index Kit with sufficient reagents for 96 samples (Catalog # FC-121-1011) and a 96 Index Kit with sufficient reagents for 384 samples (Catalog # FC-121-1012).
  • Can I run MiSeq Reporter on a computer independent of the MiSeq?

    You can install a second licensed copy of the MiSeq Reporter on another computer. However, that computer must be connected to the same network as your MiSeq or the network location of the output folder. The computer must meet the following minimum hardware and software requirements: 64-bit PC with at least 8GB RAM (16-32 GB RAM for optimal performance), Windows Vista or Windows 7, and at least 1 TB of available hard disk space.

  • Where is the information in the Classification Database stored?

    By default, the Classification Database is saved locally in C:\ProgramData\Illumina\Illumina VariantStudio\ClasificationDb.bin. When the database is stored locally, classifications are available for the current project and any future projects opened on that computer. The Classification Database can also be configured to store on a user-specified network location. If the classification database is stored on a network location, classifications are available to projects opened in any installation of VariantStudio with access to that network location.

  • How many samples can be processed per TruSeq ChIP Sample Prep Kit?

    Each TruSeq ChIP Sample Prep kit contains enough reagents and adapters to process 48 samples. Set A and Set B each contain 12 unique indexes, with enough of each index sufficient for 8 individual samples.

  • How can I calculate required coverage?
  • I got a warning message in HCS about the ARM9BoardSerialPort. What does this mean and what should I do?

    The warning message "ARM9BoardSerialPort (ARM9CHEM): timed out waiting" indicates that an ARM9 communication time out has occurred. The ARM9 board is one of many components that communicate between the HiSeq and instrument computer.  Messages related to an ARM9 time out are not necessarily indicative of a hardware issue, and do not impact the run or data quality.

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

  • How many uses can you obtain from the index kits?
    The index kits are good for four uses. The 96-index kit has enough reagents for 384 samples; the 24-index kit has enough reagents for 96 samples. Additional caps are included for the tubes to replace after each use in order to prevent cross-contamination.
  • What do I need to run MiSeq v3 chemistry?
    Using reagents provided in the MiSeq Reagent Kit v3 requires the recipes and RFID recognition settings in MiSeq Control Software (MCS) v2.3. To download a copy of MCS v2.0, see the MiSeq System downloads page.
  • What is the recommended PhiX spike in?

    Illumina recommends the following PhiX spike in percentage based on instrument.

    Instrument

    % PhiX Spike In
    (Greater or equal to)

     

    MiniSeq

    10%

     

    MiSeq

    5%

     

    NextSeq

    10%

     

    HiSeq 2500

    5%

     

    HiSeq 3000/4000
    using RTA v2.7.6 or earlier

    20%

     

    HiSeq 3000/4000
    using RTA v2.7.7 or later

    5%

     

  • What TruSight One kits are available and how many samples can be processed with each kit?

    Catalog Number

    Kit Name

    FC-141-1006

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

    FC-141-1007

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

    TG-141-1006

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

    TG-141-1007

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

  • How many samples can I sequence at a time on the MiniSeq System?

    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.

  • How much DNA can be targeted with this kit?

    This kit enables targeting of over ~650 kb of cumulative DNA sequence (1536 amplicons x 425 bp each = ~650 kb).

  • What is the shelf life of the kits?

    Expiration dates are printed on the kit box labels and reagent tubes. Illumina guarantees 3 months of shelf life from the time of kit shipment. 

  • What is the library size distribution for Nextera libraries?
    The Illumina Nextera DNA Sample Preparation kit produces libraries with a broad size range distribution, typically between 300-1000bp. Please refer to the Nextera DNA Sample Preparation Guide for more specific details.
  • How long does it take to perform a MiniSeq run?

    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.

  • What is the input amount required for TruSeq library prep?
    • ChIP: 5–10 ng ChIP-enriched, fragmented DNA
    • DNA: 1 μg gDNA
    • DNA PCR-Free:
      • 1 µg gDNA for a 350 bp insert size
      • 2 µg gDNA for a 550 bp insert size
    • Nano DNA:
      • 100 ng gDNA for a 350 bp insert size
      • 200 ng gDNA for a 550 bp insert size
    • RNA: 0.1-1 μg total RNA
    • Small RNA: 1 μg total RNA
    • Targeted RNA Expression:
      • 50 ng total RNA
      • 100-200 ng degraded or FFPE RNA, depending on fragment size
  • Are NovaSeq 5000/6000 flow cells available in single-read and paired-end versions?

    NovaSeq 5000/6000 reagent kits include all consumables to support both single-read and paired-end sequencing. During run setup, you can configure the run to generate either single-read or paired-end data.

  • Which aligner should I use in the RNA-Seq Alignment App?

    The STAR aligner in the RNA-Seq Alignment App is recommended. This aligner has been optimized for fusion calling.

    You can also use the TopHat Alignment App. It can be useful to run both analysis options and compare the data.

  • Is there a separate cost for TruSeq Synthetic Long-Read DNA data analysis?
    Purchase of the kit includes access to TruSeq Long-Read Assembly or TruSeq Phasing Analysis applications in BaseSpace.
  • What is the storage capacity of the MiSeq integrated computer?
    The integrated instrument computer has approximately 550 GB of storage capacity. For greater storage capacity, you can securely analyze, store, and share your MiSeq data using BaseSpace, Illumina's customized cloud computing environment that eliminates the need for onsite infrastructure.
  • What percentage of content from TruSeq Methyl Capture EPIC overlaps with the Infinium Human Methylation EPIC BeadChip?

    Over 90% of Infinium Human Methylation EPIC BeadChip target regions are also covered by TruSeq Methyl Capture EPIC target regions.

  • How long does it take to complete an array scan on the NextSeq 550 system?

    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.

  • Does the mRNA-Seq kit include controls?
    No specific controls are included with this kit. The PhiX sequencing control (sold separately) is necessary for calibrating the instrument for each run. For a sample-prep-level positive control, we recommend the Universal Human Reference RNA from Strategene.
  • How are images taken on the HiSeq or HiScanSQ?
    Images are taken using a time delayed integration (TDI) line scanning optical system with four CCD sensors. The TDI line scanning system greatly increases throughput by maximizing camera utilization.
  • What is the benefit of increasing the Desired Probe Spacing in targeted regions of my Nextera Rapid Capture Custom Enrichment DesignStudio project?
    Increasing the Desired Probe Spacing setting (from Standard toward Overlapping) increases the density of probes designed across the submitted target region. For most designs, this means there are more total probes within the region of interest and the likelihood of enriching that region is increased.
  • What indexing options are available on the NovaSeq System?

    NovaSeq 5000/6000 reagent kits support single and dual indexing options. The kits include sufficient reagent for 47 additional cycles: 7 chemistry-only cycles plus up to 40 cycles for the index reads. The indexing primer provides primers for both single and dual indexing. The NovaSeq System follows the same dual indexing workflow as the HiSeq 2500 system.

  • How do I ensure I have an accurate amount of input DNA for the Nextera XT kit?

    The Nextera XT DNA Sample Preparation Kit protocol is optimized for 1 ng of input DNA total. Illumina strongly recommends quantifying the starting genomic material. Nextera XT DNA Sample Preparation library preps use an enzymatic DNA fragmentation step and thus can be more sensitive to DNA input compared to mechanical fragmentation methods. The ultimate success of the assay strongly depends on using an accurately quantified amount of input DNA library. Therefore, the correct quantitation of the DNA library is essential.

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

  • What is the intensity spike after template generation?
    For HiSeq v4 runs, there might be a noticeable increase in the reported intensity by cycle in SAV after template generation has completed after cycle 5. This increase occurs at this point because the reported intensities include only the clusters included in the final template. Before template generation, total intensity is reported.
  • What is SRF?
    SRF (Single Read Format) is a generic format for DNA sequence data. The format is defined at http://srf.sf.net.
  • Are there any in-line controls in the sample prep?
    No.
  • What is the minimum gap size required between adjacent target regions in DesignStudio?

    A gap equivalent to a maximum amplicon size is required between target regions; regions with less than this gap size are merged to improve design performance and prevent unfavorable probe to probe interactions. The following table shows the maximum and minimum amplicon lengths for a given amplicon size setting.

    Setting

    Minimum

    Maximum

    150

    125

    190

    175

    170

    190

    250

    225

    275

    425

    400

    450

  • What is the plan for legacy Illumina sample prep kits?
    • mRNA-Seq Sample Prep Kits - available until the end of 2011.
    • Genomic and Paired-End Sample Preparation Kits are currently available for order. Customers will be notified of any changes to the availability of these kits.
    • Small RNA Sample Preparation Kits - available until the end of 2011.
    • Genomic, Paired-End, Multiplexing Oligo Only Kits are currently available for order. Customers will be notified of any changes to the availability of these kits.
  • How many cycles are required for template generation on the NextSeq system?

    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.

  • Can HiSeq v2 and TruSeq rapid SBS reagents be combined?

    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.

  • What is the difference between the TruSeq DNA PCR-Free LT and HT Library Prep kits?

    The TruSeq DNA PCR-Free LT Library Prep Kits come with adapter index tubes recommended for preparing 24 or less samples at a time. The LT kits come in two sets, A and B, and each set contains 12 unique indices for a total of 24 unique single-index adapters if both kits A and B are combined. Each kit (LT kit A or LT kit B) come with reagents sufficient for 24 samples. The TruSeq DNA PCR-Free HT Library Prep Kit comes in a 96 well adapter plate format with 96 dual-indexed adapters and enough reagents for 96 samples.

  • I see a 130-135 bp band on my final ChIP-Seq library. What is this?

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

  • How is the disease association determined in the RNA Fusion module tables?

    The disease associations are defined by data collected from the Mitelman Database on August 26, 2015. The RNA Fusion module does not retrieve updates to the database and users cannot update the database. The RNA Fusion module reports only the most frequently observed disease association of the fusion from scientific literature recorded in the Mitelman database as of August 26, 2015. Disease associations for fusions with undefined disease associations are reported as “NA” (not available). The disease association that the RNA Fusion module provides, via the Mitelman Database, is for research use only and must not be used for any clinical decisions. The TruSight RNA Fusion System is classified as Research Use Only.

    "Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer (2015/08/26). Mitelman F, Johansson B and Mertens F (Eds.), http://cgap.nci.nih.gov/Chromosomes/Mitelman"

  • What is a manifest file and where do I get a manifest file for my project?

    Manifest files contain a list of genomic regions and coordinates being targeted for enrichment in the library prep kit. They are also required in the data analysis process for alignment and variant calling in targeted regions. Each product contains a _targeted regions and _probe manifest file.

    You can download manifest files from the Downloads support page of the library prep kit.

  • Which software should I use for downstream analysis of clusters and for generating genotype calls?

    Illumina does not provide recommendations for downstream analysis outside of GenomeStudio.

  • Does Illumina offer TruSeq Library Prep Oligo Only Kits?

    Oligo Only Kits are not offered for the TruSeq DNA and RNA Library Prep kits.

  • How long does it take to generate ready-to-sequence TruSeq RNA Access libraries?
    It takes 2 days for 8-16 samples and 3 days for 17-48 samples, from total RNA input until libraries are ready to load on the flow cell. This includes approximately 11 hours of hands-on time.
  • Do I need to make full 96-plex or 24-plex pools?
    No, lower levels of multiplexing are possible. Please see the Nextera DNA Sample Preparation Guide for more information on pooling recommendations for samples less than the full kit size.
  • What method should I use to quantify my input genomic DNA for the Nextera Rapid Capture Enrichment protocol?
    Illumina recommends using a fluorometric-based method that is specific to double-stranded DNA, such as QuantiFluor or PicoGreen. For more information, see the DNA Input Recommendations section of the Nextera Rapid Capture Enrichment Guide.
  • Where are the content manifest file downloads for my Nextera Rapid Capture Custom Enrichment assay pool?
    Target and Probe manifest files can be downloaded from the following locations online:
    • The Export Manifest function in Project Dashboard of your DesignStudio project
    • The Product Files section of in Custom Products in your MyIllumina account
  • What is the throughput of the HiSeq 4000 system?

    Each system can generate up to 1.5 Tb in 3.5 days with greater than 75% of bases above Q30 from a 2 x 150 bp run. This throughput enables up to 12 genomes at 30x per run per system. For more information, see HiSeq 3000/4000 System Specifications.

  • What are the dimensions of the NextSeq system?

    For instrument specifications, see the Site Prep/Lab Environment page.

  • How many barcode plates do I need to run to finish my TruSeq Synthetic Long-Read DNA genome?
    The Synthetic Long-Read application generates at least 600 MB of synthetic long reads (>1.5 kb). The number of barcode plates prepared depends on the target genome size and the depth of the existing build. A reasonable starting point might be 5-10x genome coverage of synthetic long reads for genome finishing.
  • What method should I use to quantify my final TruSeq Synthetic Long-Read DNA libraries?
    Quantify your libraries using a fluorometric quantification method that uses dsDNA binding dyes or qPCR.
  • Are there any installation dependencies for VariantStudio?

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

  • How do I read the TruSeq Sample Prep controls?

    RTA 1.9 (on GA) or 1.10 (on HiSeq) or higher processes the controls. To visualize them, SAV (Sequencing Analysis Viewer) 1.7 or higher must be used. RTA produces a new InterOp metrics file called ControlMetrics; this is the file SAV uses to get control counts.

  • What level of indexing is supported?

    See the following chart for the recommended number of samples per flow cell to obtain > 40x mean coverage and > 90% of target bases covered at ≥ 10x. This kit only includes indexing for up to 12 samples.

    Instrument

    Mode

    Samples/Flow Cell

    NextSeq

    High-Output

    8

    HiSeq 2500

    Rapid

    4-6

     

    High-Output
    (Samples/Lane)

    32
    (4)

    HiSeq 3000/4000


    (Samples/Lane)

    48
    (6)

     

     

     

  • Can dual-indexed libraries be combined with other library types in the same flow cell?

    Illumina supports sequencing libraries prepared by different sample prep kits such as single-indexed libraries and dual-indexed libraries in different lanes of the same flow cell. To do this, you must use a dual-indexed workflow and the appropriate dual-indexed primers, and prepare two sample sheets. For more information, see Sequencing Mixed Libraries on a HiSeq or GA Flow Cell.

  • What is TruSeq Targeted RNA Expression?

    TruSeq Targeted RNA Expression is a library prep workflow that enables efficient, multiplexed gene expression profiling for 12–1000 targets per sample and up to 384 samples in a single MiSeq run.

  • How do I assess the quality of input DNA?

    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.

  • What kit sizes are available for the NextSeq system?

    NextSeq kits are available in three sizes:

    • The high-output kit is available in300 cycles (2 x 150), 150 cycles (2 x 75), and 75 cycles (1 x 75).
    • The mid-output kit is available in 300 cycles (2 x 150), 150 cycles (2 x 75).

    For output specifications, see NextSeq 500 specifications page

  • Can I save my filter settings as a template for use with other data sets?

    Yes. After selecting filter settings using options in the Filters pane, you can save the filter for later use. To apply a saved filter, click the drop-down menu in the Current field, and select a filter name from the list. The filter is automatically applied.

  • What is a TruSeq Targeted RNA Expression add-on project?
    An add-on project uses a fixed content pool, which can be either a fixed panel or previously ordered custom panel, and allows you to add 12-1000 additional custom targets.
  • How many samples can I process at a time?

    Using the LT kit, up to 12 samples can be processed at a time.

    Using the HT kit, up to 48 samples can be processed at a time.

  • Can I perform comparisons across large groups of samples for cohort or population analysis?

    No. BaseSpace Variant Interpreter does not support comparisons of 2 or more groups of samples. Use BaseSpace Cohort Analyzer for cohort or population analysis.

  • What is the difference between the TruSeq sample prep HT and LT kits?

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

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

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

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

  • How long does the assay take?

    The assay takes less than 8 hours total, with 2–3 hours of hands-on time. The libraries are then immediately compatible with the MiSeq system without any further manipulation.

  • What method do I use to quantify my input genomic DNA for the TruSight One

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

  • What is the cutover plan for TruSeq DNA and RNA from v1 to v2/LT?
    TruSeq DNA and RNA v1 kits are no longer available for order. Contact your local Account Manager for assistance in transitioning to the latest TruSeq RNA and DNA Sample Prep Kits for sequencing projects.
  • Can I store the BeadChip adapter on the imaging compartment stage when the NextSeq 550 system is not in use?

    Yes; however, do not store the BeadChip adapter without a BeadChip on the imaging compartment stage on the NextSeq 550 system. 

  • What sequencing primers should I use for TruSeq ChIP libraries?

    TruSeq ChIP libraries are compatible with sequencing primers included in all TruSeq cluster kits as well as in TruSeq Dual Index Sequencing Primer Box, Single Read or Paired End.

  • What is the recommended starting insert size range of ChIP DNA for TruSeq ChIP sample prep?

    Illumina recommends a DNA insert size range of 200–800 bp.

  • Does Illumina curate the annotation database?

    No. Illumina provides annotations from the respective annotation sources as is.

  • Can HiSeq v4 and TruSeq v3 high output SBS reagents be combined?

    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.

  • What is included in a NeoPrep library prep kit?

    To perform a run on the NeoPrep System, a single-use NeoPrep System library prep kit is required. Each kit contains the following for a single run:

    • Assay specific reagent plate and guide affixed on the reagent plate
    • Assay specific reagent tubes
    • Oil vial and funnel
    • Library card
    • Library card guide
    • Library separation tube strips

    For specific library prep kit contents and storage conditions, see the appropriate library prep reference guide.

  • Can DNA from FFPE samples be used?
    Yes, the Cancer Panel has been optimized for use with DNA derived from FFPE sources. A simple qPCR-based quality control test (WG-321-1001, Illumina FFPE QC Kit) is recommended to ensure maximum sample success.
  • What are the differences on the GA between version 8.3, 8.4, 8.5 and version 10 recipes?

    All recipes use latest SBS chemistry but only v10 recipes enable dual indexing and eight-base barcodes for both single-index and dual-index runs.

    • The difference between 8.3 and 8.4 is the introduction of a user wait after Read 1 to load freshly made indexing reagents, which removes the need to load indexing reagents at the beginning of the run to improve index read quality due to aging of reagents.
    • The difference between v8.4 and v8.5 is that the user wait between Read 2 priming and Read 2 resynthesis has been removed. However, a user wait has been added after Read 2 resynthesis and before first base incorporation to load Read 2 SBS reagents.
    • V10 recipes have the same workflow as v8.5 recipes, but are compatible with eight-base barcodes and dual-indexed runs.
  • How long does it take from the start of the run until I have cluster density metrics?
    Depending on cluster density, metrics appear at the beginning of cycle 20, if you are running MCS v2.3. With earlier version of MCS, metrics appear at the beginning of cycle 7.
  • Why is GC high in the first few bases?
    It is normal to observe both a slight GC bias and a distinctly non-random base composition over the first 12 bases of the data. For example, you might see it in the IVC (intensity versus cycle number) plots that are part of the output of Pipeline analysis software. In genomic DNA sequencing, the base composition is usually quite uniform across all bases, but in mRNA-Seq, the base composition is noticeably uneven across the first 10 to 12 bases. We believe this effect is caused by the "not so random" nature of the random priming process used in the protocol. This might explain why there is a slight overall G/C bias in the starting positions of each read. The first 12 bases probably represent the sites that were being primed by the hexamers used in the random priming process. The first 12 bases in the random priming full-length cDNA sequencing protocol (mRNA-seq) always have IVC plots that look like what has been described. This is because the random priming is not truly random and the first 12 bases (the length of two hexamers) are biased towards sequences that prime more efficiently. This is normal and expected.
  • What Illumina software is available for TruSeq ChIP data analysis?

    CASAVA and (MSR) can be used for demultiplexing. However, they are not intended for TruSeq ChIP analysis.

  • What method is recommended to quantify my DNA for the TruSeq Rapid Exome protocol?

    To quantify input genomic DNA and DNA in the PCR and enriched libraries, use a fluorometric-based method that is specific to double-stranded DNA, such as QuantiFluor or PicoGreen. The concentration of gDNA can be determined using the Qubit dsDNA BR Assay or the Qubit dsDNA HS assay. These assays use a fluorescent dye that is highly selective for double-stranded DNA over RNA and can detect samples in a concentration range from 10 pg/μl – 1000 ng/ μl. PicoGreen dye can also be used to accurately measure the DNA concentration.

    For more information, see the DNA Input Recommendations section of the TruSeq Rapid Exome Reference Guide.

  • Can I expect comparable library preparation performance between the TruSeq LT and HT kits?

    There are variances between the TruSeq LT and HT kits that may result in differences in yield and the amount of adapter dimer for the final library. Be sure to take extra care pipetting the AMPure XP beads accurately in the Clean Up PCR step, to minimize the amount of adapter dimer carried through to the final library. Also, note that the AMPure XP bead ratio at this step is optimized for the longer length of the dual-index adapters.

  • Where do I load the 8-port and 10-port gaskets with a 2-lane rapid flow cell and an 8-lane high output flow cell?

    For setting up either a Rapid Run mode, TruSeq v3 mode, or HiSeq v4 mode on a HiSeq 2500 or HiSeq 1500, load the 8-port gasket in the back manifold position and load the 10-port gasket in the front manifold position. For a run on a HiSeq 2000, load 8-port gaskets in both the front and back positions.

    If you use the TruSeq Cluster Kit v3 and need 10-port gaskets for the front manifold, contact Illumina Technical Support.

  • How does this protocol handle ribosomal RNA contamination?

    Due to the enrichment step in the workflow, the ribosomal RNA is washed away during the hybridization and capture steps. The hybridization times have been optimized to allow for less rRNA to be captured during the enrichment pull-down steps. The residual amount of ribosomal RNA contamination can be determined from the “% Aligned to ribosomal RNA” field in the sample analysis report.

  • How does the array data quality on the NextSeq 550 system compare to an iScan system?

    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.

  • How can I tell which genome my sequences were aligned to?
    See the CASAVA User Guide, available in the GenomeStudio Portal.
  • How would I enable analysis of both single and dual index samples in the same flow cell using CASAVA 1.8.2?

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

  • Are there any variations to the protocol when using panels of different amplicon plexities and DNA inputs?

    Yes. The number of PCR cycles has been optimized for various assay conditions taking into account both the number of amplicons per reaction 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.

  • When is analysis of a sequencing run performed?
    Image analysis occurs in real time, phasing estimates and base calling begin occur after cycle 12, and base call quality scoring occurs after cycle 25.
  • Have different levels of DNA fragmentation been tested with this kit?
    Customers can use the Illumina FFPE QC Kit (WG-321-1001) to determine whether their samples meet the minimum quality criteria for inclusion in a Cancer Panel experiment. This protocol is qPCR based, where a Ct (threshold cycle) cut-off allows users to determine whether their samples will succeed in the TruSeq Amplicon assay or not. This measurement represents how well DNA amplifies and can be used to infer DNA quality. Testing has demonstrated that over 80% of samples passing the Illumina FFPE QC Kit protocol are expected to succeed in the TruSeq Amplicon assay.
  • Are there compatibility requirements for using the NextSeq 500/550 Kit v2?

    Yes. Using the NextSeq 500/550 Kit v2 requires NextSeq Control Software (NCS) v1.4.

  • Can Nextera dual-indexed libraries be combined with other library types on the same flow cell?
    Yes. If you are sequencing combined single-indexed and dual-indexed libraries on the HiSeq or GA, use sequencing primers provided in the TruSeq Dual Index Sequencing Primer Box (Single Read, Catalog # FC-121-1003 or Paired End, Catalog # PE-121-1003). Primers provided in this kit are compatible with all library types. Single-indexed libraries and dual-indexed libraries need to be loaded onto separate lanes of the flow cell. For more information, see Sequencing Mixed Libraries on a HiSeq or GA Flow Cell.
  • Can Nextera dual-indexed libraries be combined with other library types on the same flow cell?
    Yes. If you are sequencing combined single-indexed and dual-indexed libraries on the HiSeq or GA, use sequencing primers provided in the TruSeq Dual Index Sequencing Primer Box (Single Read, Catalog # FC-121-1003 or Paired End, Catalog # PE-121-1003). Primers provided in this kit are compatible with all library types. Single-indexed libraries and dual-indexed libraries need to be loaded onto separate lanes of the flow cell. For more information, see Sequencing Mixed Libraries on a HiSeq or GA Flow Cell.
  • What is the general workflow for designing a Nextera Custom Enrichment project in DesignStudio?
    • Choose a project name and details (genome builds, etc)
    • Submit targets with source and build
    • Choose exons or full regions
    • Design is automatically executed
    • Visualize probes
    • Choose probes to place in final design
    • Review design
    • Place order
  • What is the recommended read length for TruSeq Sample Prep?
    • TruSeq DNA Sample Prep supports up to 2 x 150 bp runs on the GA and up to 2 x 100 bp runs on the HiSeq/HiScanSQ
    • TruSeq RNA Sample Prep supports up to 2 x 75 bp runs on the GA and up to 2 x 75 bp runs on the HiSeq.  It is important to note that eland_rna will not handle paired-end mRNA runs, and RNA runs over 50 cycles will show artificially elevated error rates due to the reads crossing multiple splice junctions.  The algorithms used in CASAVA will give the best results up to 50 cycles. Third-party analysis packages may handle these reads differently.
  • Can I use the TruSeq Stranded RNA HT sample prep kits to generate libraries from FFPE samples or from only intact total RNA?
    The TruSeq Stranded mRNA HT Sample Prep Kit supports high quality RNA as input for sample prep as the protocol relies on a poly-A tail purification step. The TruSeq Stranded Total RNA HT Sample Prep Kit is compatible with FFPE samples. Please see the RNA Input Recommendations section of each kit's user guide for more information.
  • How do I install and access BaseSpace Variant Interpreter?

    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 11, or later.

  • Which workflow is required to create a sample sheet using Illumina Experiment Manager?

    To create a MiSeq-compatible sample sheet, select the RNA Sequencing category and the RNA-Seq application or the Other category and the FASTQ Only application. For the library prep kit, select TruSeq LT. Then select 1 Index Read, Paired End Read, and 76 bp Read Length.

    For more information, see the IEM TruSight RNA Pan-Cancer and TruSight RNA Fusion Quick Reference Card.

  • How does TruSeq Synthetic Long-Read DNA library prep technology work?
    The TruSeq Synthetic Long-Read DNA Library Prep and Barcode kits are designed for two applications; preparing DNA libraries for de novo synthetic long-read sequencing and analysis and phasing analysis from whole human genome sequencing data. The kits combine TruSeq and Nextera chemistries to prepare DNA libraries for sequencing and informatics apps to aid in performing long reads assembly or phasing analysis.
  • Is NaOCl required for the NextSeq instrument wash?

    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 Manual Post-Run Wash option introduced with NCS v1.4 requires diluted NaOCl.
    • The manual wash option provided with NCS v1.3 or earlier versions does not require NaOCl.
  • Is there a PCR-free TruSeq Sample Prep protocol available?

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

  • Are AMPure XP beads supplied with the kit?

    All beads required for the protocol are supplied in the kit.

  • How much input genomic DNA is required for the TruSeq Rapid Exome workflow?

    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.

  • What is the Mitelman Database?

    The Mitelman Database is a reference for common fusions.

  • What is the sensitivity for Fusion detection?

    Sensitivity of fusion detection varies depending on the expression level of the fusion transcript in the sample. We performed titration experiments with synthetic fusion constructs. Spike-in fusions were detected as low as concentrations equivalent to ~13 fusion copies/cell (1 E-7 pM) (based on 500 cells/10ng input).

    The RNA-Seq Alignment App requires a minimum of 3 unique fusion-supporting reads to call a fusion positively (with some additional quality and read metrics). However, a high number of nonfusion supporting reads in that region is expected to cause ‘noise’ that can affect fusion calling.

  • Is the TruSight One assay compatible with BaseSpace?

    Yes, BaseSpace can be used for the analysis of TruSight One runs. Illumina recommends using the Illumina Experiment Manager and select Targeted Sequencing and the subsequent Enrichment workflow.  Download VCF files from BaseSpace projects to filter variants using the VariantStudio software.

  • Can I split my 200-cycle kit for shorter runs?
    Yes. You can split the 200-cycle SBS Kit into two equal volumes suitable for up to 101 cycles each. See the TruSeq SBS Kit Reagent Preparation Guide (200 Cycles) for instructions and storage requirements. If you need smaller volumes for shorter runs, Illumina recommends using the TruSeq SBS Kit (50 Cycles).
  • Are Nextera Rapid Capture Enrichment indexes and sequencing primers the same as the Nextera and Nextera XT sample prep?

    Nextera Rapid Capture Enrichment index 1 (i7) and index 2 (i5) sequences are identical to index sequences used in other Nextera based kits. They use the Read 1 (HP10), Read 2 (HP11) and Index 1 (HP12) sequencing primers.

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

  • What method is recommended for quantifying input gDNA?

    Illumina recommends using a fluorometric based methods including Qubit or PicoGreen to provide accurate quantification. UV based methods such as NanoDrop, measures any nucleotides present in the sample including RNA, dsDNA, ssDNA and free nucleotides, which can give an inaccurate measurement.

    DNA Type

    Input

    Concentration

    A260/A280

    High-quality genomic DNA

    50 ng

    10-25 ng/µl

    1.8-2.0

  • Can I use Nextera Rapid Capture Enrichment, Nextera DNA, or Nextera XT DNA library prep kits with TruSeq Rapid Exome reagents?

    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.

  • What method is recommended to quantify my enriched library?

    Quantify the enriched library using a fluorometric-based method specific to double-stranded DNA, such as QuantiFluor or PicoGreen. For more information, see the Validate Library section of the library prep reference guide.

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

  • Can I order a 300-cycle MiSeq v3 reagent kit separately from the TruSight One sample prep kit?
    Three (3) MiSeq v3 reagent cartridges are included with the 9-sample TruSight One Sequencing Panel kit (FC-141-1006). If you need additional MiSeq reagent cartridges, it is necessary to order additional TruSight One 9-sample kits or MiSeq v3 600 cycle kits (MS-102-3003).
  • Can the Illumina Small RNA v1.5 adapters and primers be purchased separately for directional RNA sample preparation?

    No, there is currently no Small RNA v1.5 oligo-only kit available. 

  • Which flow cell can be used with these libraries?

    A paired-end flow cell from any Illumina sequencing system can be used with these libraries.

  • Can data from Illumina Nextera libraries be directly compared to legacy Nextera kits?
    The Illumina Nextera kit has been optimized and performances between kits may be different. For example, the new Illumina Nextera DNA Sample Preparation kits exhibit improved GC performance compared to the legacy kits.
  • Is the output of bcl2fastq compatible with CASAVA v1.8.2?

    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.

  • How many indexes are included in each kit?

    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.

  • Are separate pre-PCR and post-PCR areas required?

    Ideally, perform pre-PCR and post-PCR procedures in 2 separate rooms. If separate equipment is used (eg, 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.

  • Why might I see different insert sizes when trying to aim for a 350 bp or 550 bp TruSeq Nano DNA Library Prep insert size? How can I improve it?

    Changing amounts of beads or volume inaccuracies during the beads size selection step will impact the final insert sizes. New users should take the TruSeq Sample Purification Bead Size Selection and Best Practices online training course prior to performing TruSeq Nano DNA library prep. The HS protocol may provide more consistency between samples.

  • Is there a manual way to include or exclude samples from clusters?

    Yes, in the SNP graph, use the curser to draw a box around the samples you wish to manually edit, right-click and choose the cluster samples should be assigned to, or NC (no call) if you wish to remove samples from any clusters.

  • Can I run a control lane on a MiSeq flow cell?
    No, a dedicated control lane is not possible because the MiSeq flow cell a single-lane flow cell. Illumina recommends using a spike-in method for adding a control to libraries.
  • How do I change the instrument health upload option?
    From the Welcome screen, select Menu, then Tools. The Options menu includes the checkbox to turn on or off instrument health data. Select View Terms for more information about the instrument health option.
  • Do I need a cBot instrument to be able to run sequencing flow cells on my HiSeq 1500/2500?
    A cBot is needed for cluster generation on 8-lane High Output flow cells or for loading separate samples in each lane of a 2-lane Rapid flow cell. A cBot is not needed if you only plan to utilize the Rapid run HiSeq functionality and load the same sample across the entire flow cell.
  • I do not have GenomeStudio installed on my computer, but would like to work with the PC Module. Do I need to install GenomeStudio prior to installing the PC Module?

    Yes, please download the GenomeStudio 2011.1 installer from the Illumina website. It is sufficient to install the GenomeStudio Framework by clicking the respective box in the install wizard, which does not require a license key. It is not required to install the GenomeStudio Genotyping Module on the same computer on which the PC Module is installed. However, the polyploid workflow does require generating a genotyping project in the GenomeStudio Genotyping Module prior to taking the data to the PC module for polyploidy clustering. 

  • What is the shelf life for NuPCR reagents?
    The shelflife of NuPCR Assays and Master Mix is 12 months from the date of manufacture when stored at -15° to -25°C .
  • How do I select the right number of index cycles and chemistry for sequencing dual-indexed libraries in HCS 1.5?

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

  • Does Illumina VariantStudio support the genome VCF (gVCF) format?

    Yes. VariantStudio v2.1 and v2.2 support VCF and gVCF file formats. For more information, see the VariantStudio User Guide.

  • Can I modify the classification categories in VariantStudio?

    Yes. By default, VariantStudio provides five classification categories: Pathogenic, Presumed Pathogenic, Unknown Significance, Likely Benign, and Benign. You can edit these categories through the Annotation & Classification | Classification Settings menu. 

  • What sample prep do I use for MiSeq?

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

  • How do I analyze data from a run on the MiSeq?

    The system is designed to support multiple workflows inclusive of data analysis, which is performed on-instrument upon completion of the run. Output file formats are *.bcl, FASTQ, BAM, *.vcf, *.csv, and *.txt.

  • Can I use custom primers on the NovaSeq System?

    Yes, the NovaSeq 5000/6000 Cluster Cartridge and the NovaSeq Control Software allow the use of custom primers for Read 1, Read 2, and the Index 1 Read. Positions 5, 6, and 7 on the cluster cartridge are reserved for custom primers and the control software includes an option to select custom primers during run setup.

  • What applications are recommended for the Nextera XT kit?
    The Nextera XT kit is recommended for small genomes, PCR amplicons greater than 300 bp, plasmids, microbial genomes, concatenated amplicons, and double-stranded cDNA.
  • How do I connect to LIMS?

    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).

  • What Bionanalyzer chips are recommended to validate the final TruSeq Nano DNA library and what are the recommended dilutions?
    The protocol recommends using the DNA 7500 chip with no dilution or the High Sensitivity chip with a 100 fold dilution. DNA 1000 chip should not be used as the upper marker may overlap with the library peak.
  • What forms of sequencing-based methylation are there and which do you recommend?

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

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

       

  • How do I set up the sample sheet for MSR analysis?

    Use the Enrichment workflow to create a sample sheet formatted for analysis of TruSight One data. Refer to the IEM TruSight One or TruSight Rapid Capture Quick Reference Card for instructions on creating your sample sheet.

  • What is the difference between the Nextera Rapid Capture Exome and Expanded Exome Enrichment?

    The Nextera Rapid Capture Expanded Exome Enrichment utilizes the same capture oligos as the TruSeq Exome Enrichment Kit paired with the new Nextera Rapid Capture Enrichment reagents.

     

    Exome

    Expanded Exome

    Targeted Region

    ~45 Mb

    ~62 Mb

    Content

     

    Coding Exons Exons, UTR, miRNA
  • What Nextera Enrichment kits are available?
    Two different Nextera Enrichment kits are available: an Exome Enrichment kit and a Custom Enrichment kit. Both kits utilize the fast, simple, high throughput workflow of the Nextera technology in combination with a comprehensive enrichment solution. Nextera Exome Enrichment offers 24 indices for enriching up to 48 samples at 12-plex (catalog # FC-121-1204) or 96 samples at 12-plex (catalog # FC-121-1208). Nextera Custom Enrichment includes 14 indexes that create 24 barcode combinations for enriching up to 48 samples at 12-plex (catalog # FC-123-1204) or 96 samples at 12-plex (catalog # FC-123-1208) and 20 indexes that create 96 barcode combinations for enriching up to 288 samples at 12-plex (catalog # FC-123-1224).
  • What is the difference between the BaseSpace RNA-Seq Alignment and TopHat Alignment apps? Which app should I run?

    The main difference between the apps is that the RNA-Seq Alignment App is specifically designed for optimal fusion calling using STAR.

    You can try both analysis methods and compare results.

  • How do I deselect MiSeq Reporter when I start a run?

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

  • What makes a magnet suboptimal for this library prep?

    Two major aspects can make a magnet suboptimal for this protocol:

    1. If the magnet is too weak and does not sufficiently pull beads out of solution.

    2. If the geometry of the magnet makes it difficult to remove the entire supernatant cleanly, without removing any beads along with the supernatant.

  • How much free space on the NextSeq 550 system hard drive is required for array scanning?

    Array scanning requires about 400 MB of free space on the NextSeq 550 hard drive. NCS v2.0 automatically manages disk space.

  • How is the Illumina technology different from the CE-based technology for ForenSeq DNA Signature Prep?

    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.

  • What are CBCL files?

    CBCL files (*.cbcl) are concatenated BCL files that save space and optimize output. Instead of generating BCL files on a per tile basis, tiles from the same lane and surface are aggregated into 1 CBCL file for each lane and surface.

  • What flow cells and reagents are supported on the HiSeq 3000?

    Reagents and flow cells provided in the HiSeq 3000/4000 SBS Kit and the HiSeq 3000/4000 Cluster Kits are designed and optimized for the HiSeq 4000 and HiSeq 3000 systems. Reagent kits designed for other HiSeq systems are not compatible with the HiSeq 4000 and HiSeq 3000 systems.

  • When generating fewer than the full amount of samples supported by a TruSeq HT library prep kit, how should the core reagents be aliquoted or freeze/thawed?

    The HT kit provides sufficient reagents for 96 samples, in two tubes for 48 samples each. If you do not intend to consume the entire reagent in a tube in one use, we recommend dispensing the reagent into aliquots (a minimum of 24 sample preps/aliquot) after the initial thaw, and refreeze the remaining aliquots to avoid excessive freeze/thaw cycles. However, if you aliquot, you may not have enough reagents for the full number of reactions over multiple uses. When preparing less than 24 samples at a time, Illumina strongly recommends using the LT kit.

  • How do I analyze my data?

    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.

  • How long can a rapid flow cell be stored after finishing template hybridization and first extension on the cBot and before being run on the HiSeq 2500?

    You can store the rapid flow cell up to 24 hours after template hybridization and first extension. However, Illumina recommends that you start the sequencing run on the same day.

  • Are there in-line controls included in the small RNA kit?

    There are no in-line controls for the small RNA kit.

  • What DNA input amount is required?

    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).

  • When using HiSeq v4 reagents, do you have to change reagents during the run as with TruSeq v3 reagents?
    HiSeq v4 kits are designed for a walk-away workflow, which means that all reagents are loaded before starting the run. Splitting incorporation buffer for Read 2 is no longer necessary.
  • Which MiSeq workflow should I choose when creating a sample sheet in Illumina Experiment Manager (IEM) for TruSeq ChIP samples?

    For MiSeq runs, a sample sheet is required at the start of the run to enable analysis. You can choose either the "FASTQ Only" or the "ChIP-Seq" application in the "Other" category when creating a sample sheet in IEM. Both workflows will generate fastq files as data output.

  • What are the recommended cluster density, output, and data quality from systems enabled for HiSeq v4?

    See the Cluster Densities Specifications technical bulletin on MyIllumina for recommendations.

  • How much total RNA input is required for this protocol?

    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.

    Quality

    DV200

    Input Requirement Per Reaction

    High

    > 70%

    20 ng

    Medium

    50-70%

    Truseq RNA Access Library Prep: 20-40 ng

    Low

    30-50%

    Truseq RNA Access Library Prep: 40-100 ng

    Too degraded

    < 30%

    Not recommended

    For more information, see the Evaluating RNA Quality from FFPE Samples tech note.

  • What percent duplicates is expected?

    The expected range of % duplicates is ≤ 25% for controls (UHR) at 0.5M subsampled reads.

  • Can I thaw the reagent cartridge in the refrigerator overnight?
    For best results, thaw the reagent cartridge in a room temperature water bath for approximately 60 minutes.
  • Are there any protocol differences based on different amplicon lengths?
    For amplicons greater than 500bp, Illumina recommends using a 0.6x AMPure XP cleanup (30 μl volume of beads). For amplicons less than 500bp, Illumina recommends using a 1.8x AMPure XP cleanup (90 μl volume of beads) to maximize yield.
  • What are the sizes of data files from the HiSeq?

    For a dual flow cell 2x101 cycle run (200 Gb) on the HiSeq 2000 using HCS v1.3 and prior, you can expect 2 TB of intensity data (optionally transferred to a server), 250 GB of base call and quality score information, and 1.2 TB of space for alignment output not including 6 TB of disk space used for temporary files removed before completion of alignment. Using HCS v1.4 and Flow Cell v3, storage requirements for raw data are approximately 60% greater than current runs based on additional swath data and increased cluster density.

  • What are the catalog numbers for the HiSeq Rapid v2 reagent kits?
    • HiSeq Rapid SBS Kit v2 - HS (50 cycle) - Cat # FC-402-4022
    • HiSeq Rapid SBS Kit v2 - HS (200 cycle) - Cat # FC-402-4021
    • HiSeq Rapid SBS Kit v2 - HS (500 cycle) - Cat # FC-402-4023
    • HiSeq Rapid PE Cluster Kit v2 - Cat # PE-402-4002
    • HiSeq Rapid SR Cluster Kit v2 - Cat # GD-402-4002
    • HiSeq Rapid Duo cBot Sample Loading Kit - Cat # CT-403-2001
    • HiSeq Rapid Rehybridization Kit - Cat # GD-404-1001
  • What indexing strategy is used in these libraries?

    The kits include 12 different Index 1 (i7) adapters (N701–N712) and up to 8 different Index 2 (i5) adapters (E501–E508), depending on the kit you are using. These libraries use the same dual 8 bp indexes as other Nextera based kits. For more information on dual index principles and sample pooling, see the library prep reference guide.

  • Can I import VCF files produced by non-Illumina software products into BaseSpace Variant Interpreter?

    Yes, the software accepts VCF files produced by the Genome Analysis Toolkit (GATK) v1.6.

  • Are MiSeq v3 kits compatible with all Illumina sample prep kits?

    Yes. Just like v2 kits, MiSeq v3 kits are compatible with all Illumina sample prep kits. Some sample prep protocols require slight changes to reach optimum clusters. A concentration of 4 nM is recommended to begin the denature and dilute step before loading libraries for sequencing. Check the Illumina Tech Support Bulletin Board for the latest information on updates to sample prep protocols.

  • Can my cBot cluster both HiSeq and Genome Analyzer flow cells?
    Yes. The cBot can cluster both HiSeq and Genome Analyzer flow cells. However, you must use the appropriate flow cell adapter plate to accommodate the different dimensions of the flow cells. Dedicating a cBot to one platform avoids routinely switching adapter plates.
  • What is the output of MiSeq?

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

  • Can I use this protocol on FFPE samples?

    Currently, this protocol has not been validated with FFPE samples

  • What are the effects of putting too much or too little genomic DNA into the Nextera Rapid Capture Enrichment protocol?
    Overloading the Nextera tagmentation reaction with more than 50 ng of genomic DNA leads to a larger library size distribution which can lower the number of on-target sequencing reads. Less than 50 ng of input DNA leads to a smaller library size distribution and reduced library diversity or elevated duplicate reads, because many of these smaller library sized fragments would be eliminated during the size-selection procedures.
  • What is the DNA input for the Nextera Enrichment kit?
    For the Nextera Enrichment kits, 50 ng of gDNA is required for input to the prep. Please note that to obtain an accurate quantification of the input DNA, Illumina recommends quantifying the starting DNA library using a fluorometric based method specific for duplex DNA such as the Qubit dsDNA BR Assay system. Upon completion of the prep, 500 ng is used as input for the enrichment portion of the protocol. Illumina recommends quantifying by picogreen or the Qubit Fluorometric Quantitation system. Please note that adding greater than 500 ng of library DNA into the enrichment assay (up to 1 ug per sample) may produce greater mean coverage per sample.
  • How many times can I add additional content to a previously ordered Nextera Rapid Capture Custom Enrichment panel?
    Additional target regions can be added to exiting content panels as many times as you like.
  • What are the input requirements for this kit?
    For the best results, use 1–10 ug. However, we have successfully used as little as 0.1 µg of UHR Total-RNA when it was highly pure and high quality.
  • What can I do about clumped magnetic beads?

    Magnetic beads can clump after the probe-hybridized RNA sample is added. This happens because the probes contain biotin that can bind to multiple magnetic bead particles. Make sure that you vortext the beads vigorously for at least 10 seconds.

  • Why are some targets difficult to design in DesignStudio?

    Homologs: Having homologs in the same design can lead to low designability. Split homologs into separate CAT pools.

    GC Content: Regions with greater than 80% GC content can be difficult to design against, particularly when these regions are greater than 500 bp in length.

    Homopolymer sequences and Repetitive elements: DesignStudio avoids these regions to make sure that probes have better specificity in the genome.

    Poor Specificity: DesignStudio will assess the specificity of probes and exclude those which will not provide satisfactory on-target coverage.

  • Can I run TruSeq Synthetic Long-Read DNA libraries on any Illumina sequencer?
    Illumina currently supports sequencing of TruSeq Synthetic Long-Read DNA libraries on the HiSeq 1000, 2000, 1500, and 2500.
  • Are my saved projects from VariantStudio v2.1 compatible with VariantStudio v2.2?

    Yes, VariantStudio v2.1projects are compatible with VariantStudio v2.2. Samples, annotations, including any imported custom annotations, and filter favorites are all carried over to VariantStudio v2.2.

  • Is a PhiX control included in the TruSeq Cluster Kit?
    No. The Illumina PhiX Control v3 is a separate product. The catalog number is: FC-110-3001.
  • How can I check whether the copying of files proceeds fast enough?

    Do the following to ensure that your network is performing fast enough for a particular run:

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

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

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

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

  • How long does it take to generate ready-to-sequence Nextera Rapid Capture Enrichment libraries?

    It takes 1.5-2 days (approximately 30 hours) from genomic DNA input until libraries are ready to load on the flow cell, including approximately 5 hours of hands on time. Nextera Rapid Capture Enrichment libraries can be loaded on the flow cell the afternoon of day 2 of the protocol.

    NexteraRapidCaptureTimeline

  • What is the typical library size distribution after Covaris shearing?

    The expected peak should be between 150-170 bp and the distribution should be >60% of the DNA between 100-300bp, and the average size of the DNA in that size range should be 180-200 bp. See the reference guide for an example of a Bioanalyzer trace.  

  • What is the length of the oligo probes used in Nextera Rapid Capture Custom Enrichment reactions?
    The read length is 80 bases.
  • Can a run be paused while the MiSeq is sequencing?
    Yes, a run can be paused briefly. The MiSeq Control Software (MCS) will pause the run and place the flow cell in a safe state. When you resume the run, the software resumes from the point that it was paused.
  • The TruSeq Nano DNA Library Prep Kit does not include in-line controls. What positive control can be used instead?

    Illumina recommends using Coriell Human-1 DNA (NA18507) or Promega Human Genomic DNA (G3041) as a positive control for this protocol.

  • What software do I use to analyze .cif files generated with HCS 2.x?

    Where .cif files can be generated, you can use OLB 1.9.4.

  • Can thumbnail images be reanalyzed on the HiSeq or HiScanSQ?
    No. Thumbnail images are for visual inspection only to help diagnose problems with a run. They are not suitable for reanalysis.
  • What sequencing, and indexing reagent combinations are compatible with the TruSeq Nano DNA Library Prep kits?

    Dual index sequencing of TruSeq Nano DNA HT libraries on the HiSeq, HiScanSQ, or GAIIx system using single-read flow cells requires the TruSeq Dual Index Sequencing Primer Box, Single Read (FC-121-1003) to be ordered separately. This add-on box is not required if sequencing a TruSeq Nano DNA HT prepared library with the MiSeq System, HiSeq Rapid two-lane single or paired-end flow cells, or on traditional paired-end flow cells for HiSeq, HiScanSQ, GAIIx. Additional SBS reagents may be necessary to account for all index cycles.

  • How large is the gap size between the Upstream Locus Specific-Oligo and the Downstream Locus Specific-Oligo for TruSeq Targeted RNA Expression?
    On average, the gap size is 15 bases, but can be as small as 1 and as large as 43 bases. The average insert size is 67 bp. The average length of the ULSO and DLSO are 25 bases, not including the PCR primer binding sites.
  • Are there any variations to the TruSight DNA Amplicon protocol when using panels of different amplicon plexities?
    Yes. The number of PCR cycles has been optimized for the number of amplicons per reaction. The recommended number of PCR cycles for different amplicon numbers is indicated in the user guide and will ensure sufficient yield for the bead-based normalization while avoiding over-amplification.
  • How is flow cell clustering conducted on MiSeq?

    Cluster generation follows the same process that occurs on the cBot in preparation for runs on the HiSeq or Genome Analyzer, except on MiSeq all reagents for cluster generation, sequencing, and paired-end chemistry are loaded onto the instrument in a pre-filled reagent cartridge prior to starting the run. When the run is started, the MiSeq performs cluster generation followed by sequencing and paired end chemistry (if applicable).

  • Can I use an original cBot manifold on the cBot 2?

    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.

  • What is the typical library size distribution for TruSeq Rapid Exome libraries?

    TruSeq Rapid Exome libraries generally range from about 200–500 bp with the main peak at about 300–350 bp. 

  • How do I analyze my TruSeq Exome data?

    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).

  • Is DNA fragmentation (i.e., with Covaris) necessary?
    No. The tagmentation step in the Nextera protocol eliminates the need for mechanical fragmentation/shearing.
  • How do data compression options in HCS v2.2 change data analysis or data handling?

    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.

  • What is the recommended minimum number of reads per sample for accurate fusion detection?

    Illumina recommends 3 million reads per sample. More or less reads may be required depending on the expression level of the fusion.

  • What read length is recommended for the sequencing of TruSight DNA Amplicon libraries?
    A 2×150 bp paired-end read is recommended for TruSight DNA Amplicon libraries such as TruSight Myeloid.
  • Does Illumina offer a custom designed TruSeq Methyl Capture EPIC panel?

    Currently, Illumina does not offer a custom designed TruSeq Methyl Capture EPIC panel.

  • Do I need to use a cBot with the MiSeq?

    No. Cluster generation is performed on the MiSeq instrument as part of the run. No other instruments are required for sequencing on the MiSeq.

  • What is the recommended starting insert size range?

    Determining the size of genomic DNA is difficult due to its large size.  However, Illumina recommends checking for DNA quality and only using gDNA with 260/280 ratio between 1.8-2.0.

  • Can these libraries be run on the MiSeq System?

    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.

  • Do I have to use a g-TUBE for TruSeq Synthetic Long-Read DNA library prep if my DNA is already fragmented?
    The protocol requires starting with high molecular weight DNA. Starting with fragmented DNA or skipping the fragmentation procedure in the protocol will lead to poor results.
  • How long does it take to ship Nextera Rapid Capture Custom Enrichment reagents after placing the order in DesignStudio?
    Typically, it takes 3–4 weeks to manufacture and ship a Nextera Rapid Capture Custom Enrichment Kit order.
  • Do I need to cluster all SNPs in the data set by the same algorithm and clustering options?

    No, you can use different algorithms and clustering options for different SNPs. The goal is to find optimal parameters for each SNP matching the biology of your samples.

  • Can I cluster a GA flow cell on the cBot 2?

    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).

  • Which metrics can be used to evaluate data in the Samples Table to identify poorly performing samples that should be excluded from the data?

    Samples can be evaluated and sorted by Poly Call Rate, Poly 10%, and Poly 50%. We recommend using the scatterplot function within the Samples Table to plot Poly 50% against Poly Call Rate to graphically visualize sample outliers.

  • What is the major difference between the DBSCAN and OPTICS algorithm, and how do I choose between them?

    OPTICS is an acronym for "Ordering Points to Identify Clustering Structure". It is a sub-algorithm of DBSCAN, developed to be more robust to changes in input parameters. This trait makes OPTICS more suited for initial clustering. DBSCAN is an acronym for "Density-based Spatial Clustering of Applications with Noise". This algorithm is more sensitive to initial input parameters such as cluster distance. DBSCAN is more suited for differentiating clusters that are very close together, and should typically applied to SNPs for which OPTICS does not yield satisfactory results. 

  • What programs can I install on the HiSeq instrument computer?

    The instrument computer is a computational engine performing real time analysis of data. To avoid loss of data and other adverse effects, Illumina does not recommend installing any additional software with the exception of anti-virus software.

  • If I continue to use VariantStudio v2.1, do I get the updated Illumina Annotation Service?

    No. VariantStudio v2.1 is programmed to use annotations from the previous version of the Illumina Annotation Service. It does not connect to the new version that is available with VariantStudio v2.2.  If you want to use the new version of the Illumina Annotation Service, use VariantStudio v2.2 and reannotate any samples in projects from VariantStudio v2.1.

  • Which genes are targeted in this panel?

    This panel targets 1385 cancer-associated genes, including 507 genes involved in fusions and > 850 genes either mutated or deregulated in cancers.

  • What method is recommended for quantifying starting material?

    Illumina recommends using fluorometric based methods for quantification, including Qubit or PicoGreen to provide accurate quantification of the gDNA. UV-spec based methods, such as the NanoDrop, measure any nucleotides present in the sample including RNA, dsDNA, ssDNA, and free nucleotides, which can give an inaccurate measurement for this kit.

  • Where do I find adapter and index sequences?

    Index sequences are located in the Appendix of the reference guide and in the Illumina Adapter Sequences Document.

  • Are separate pre-PCR and post-PCR areas required for the TruSight DNA Amplicon assay?
    Pre-PCR and post-PCR areas should ideally be in two separate rooms. If separate equipment is used (e.g., shaker, heat block, centrifuge) and a strict cleaning schedule is maintained, then TruSight DNA Amplicon can be performed in designated, non-adjacent areas in the same room. Regular bleaching is outlined in the User Guide. In addition, filtered pipette tips are also recommended.
  • Are TruSeq Rapid Exome indexes and sequencing primers the same as the ones in the Nextera and Nextera XT library prep kits?

    TruSeq Rapid Exome indexes and sequencing primers are not shared with Nextera DNA and Nextera XT library prep kits. However TruSeq Rapid Exome indexes and sequencing primers are shared with Nextera Rapid Capture library prep kits.

  • How much removal solution do I use?

    The appropriate volume of removal solution depends on the amount of total RNA input. Accurately quantify the amount of RNA and see the kit refence guide for instructions on determining the correct volume of solution.

  • Are the index sequences for the first 7 cycles of index 1 the same as current TruSeq Kits, TruSeq Custom Amplicon Kits, etc.?
    No. For specific index sequences, please refer to the Illumina Experiment Manager (IEM) or the Illumina Nextera DNA Sample Preparation Guide.
  • Can I upgrade my cBot to a cBot 2?

    No. The cBot 2 was specifically designed to provide positive sample tracking.

  • How do I dispose of NeoPrep reagents and consumables?

    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.

  • How is the workflow different from the TruSeq RNA Access workflow?

    This protocol uses single-plex enrichment, whereas TruSeq RNA Access uses 4-plex enrichment. This protocol uses single-plex enrichment to ensure even representation for all samples.

    Additionally, this protocol uses 14 PCR cycles during the enrichment step, whereas TruSeq RNA Access uses 10 cycles.

  • Are the recipes used on the HiSeq the same as on the Genome Analyzer?
    No. Recipes used with HiSeq Control Software (HCS) are unique to HCS. Likewise, recipes used with the Genome Analyzer Sequencing Control Software (SCS) are unique to SCS.
  • Can degraded or contaminated samples be used with the ForenSeq DNA Signature Prep Kit?

    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.

  • Does Illumina provide nebulizers in the TruSeq DNA Sample Prep kits?
    Nebulizers are not provided, but can be purchased separately through iCom.
  • Do I need both genes to be targeted to detect a fusion gene?

    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. 

  • Are TruSeq Custom Amplicon & TruSeq Amplicon Cancer Panel, Nextera XT applications supported on the HiSeq 2500/1500?

    Sequencing runs of these library types are compatible with rapid run reagent kits on a HiSeq 2500 in rapid run mode. However, Illumina does not currently provide software for analyzing this type of data.

  • Do you have protocols available for whole-genome or reduced-representation bisulfite sequencing?
    Yes, we have protocols available for whole-genome bisulfite sequencing (methylC-Seq), as well as reduced representation bisulfite sequencing. Note that these are Illumina-demonstrated protocols and are offered with associated restrictions and conditions. Protocols for other methods are available through third-party publications. Examples are included on our website.
  • What applications have supported workflows on the MiSeq?

    The MiSeq supports a large portfolio of sequencing applications. See the MiSeq Applications page for more information.

  • How can I tell if my genes of interest are present in the TruSight One Sequencing Panel kit?
    The TruSight One *.bed file details all regions of interest (ROI) and lists all targets of enrichment for this assay. Filter by gene name (HGNC nomenclature), or reference the coordinates of your loci of interest in the human genome reference (hg19 build). The gene list is also available on the product page (www.illumina.com/products/trusight-one-sequencing-panel.ilmn).
  • Do you recommend a bisulfite conversion kit?
    Illumina recommends the Qiagen EpiTech® Bisulfite Kit, PN 59104.
  • How does changing Cluster Distance in the Clustering Options dialog box affect my results?

    Cluster distance specifies the maximum distance that samples can be away from each other and still considered part of the same cluster. Increasing cluster distance will result in fewer clusters that are larger in size, while decreasing cluster distance will result in more clusters which are smaller in size. A cluster distance of 0.06 is typically a good starting point for initial clustering.

  • What cluster generation and sequencing reagents do I use for MiSeq?

    The MiSeq System uses the MiSeq Reagent Kit, which includes specially designed and packaged reagents for cluster generation and sequencing.

  • What quality control is included in ForenSeq DNA Signature Prep?

    The ForenSeq DNA Signature Prep Kit includes two positive controls: an assay control, which is processed with the samples through library preparation, and a sequencing control, which is a prepared library included with the pooled samples before sequencing. Two negative controls can also be included: a reagent blank, which is included from the DNA extraction step, and a PCR blank, which is a nuclease-free water sample processed through library preparation.

  • What is a MiSeq manifest folder?

    The manifest folder contains manifest files required for the Custom Amplicon and PCR Amplicon workflows. A manifest file is required to specify the alignments to a reference and the targeted reference regions used in the workflow. You can specify the location of the manifest folder using the MiSeq software interface. Before you begin the sequencing run, copy the manifest file to the specified location.

  • How am I notified when a new version of BaseSpace Variant Interpreter is released?

    All registered users receive an email notification when a new software version is available. Details of changes between versions are provided in the release notes.

  • How do I analyze my data for bisulfite sequencing applications?

    Illumina suggests using a third-party methylation analysis solution such as Bismark, BSMap, or BS Seeker. See the Methylation Software section for more details.

  • How many indices are available with the TruSeq LT and HT kits?

    LT kits: 12 unique single indices in Set A and 12 unique single indices in Set B

    HT kit: 96 unique dual indices

  • How long does the cluster generation step take on the NextSeq system?

    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.

  • Can I run libraries prepared for the HiSeq or Genome Analyzer on the MiSeq?

    Yes. You can run any library on the MiSeq provided your library has complementary adapters.

  • What is that “clicking” sound coming from the HiSeq or HiScanSQ?

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

  • What level of multiplexing is supported on the MiSeq with Nextera?

    There are 96 available barcodes using 12x8 indices, combining 12 Index 1(i7) with 8 Index 2 (i5) indices.

  • How long is each index read for TruSeq HT dual-indexed libraries?

    Both Index 1 (i7) and Index 2 (i5) are each 8 bp in length.

  • How long is each index read for TruSeq HT dual-indexed libraries?

    Both Index 1 (i7) and Index 2 (i5) are each 8 bp in length.

  • What is the typical run folder size?

    The run folder size for the maximum read length of 2 x 150 is about 0.6 TB.

  • What can impact the performance of the sample enrichment?
    This is difficult to predict, but the complexity of the custom oligo library may yield lower enrichment rates and lower coverage uniformity. Additionally, low complexity libraries (e.g. less than 2 Mb capture) or high GC content areas may have lower enrichment or coverage uniformity as well.
  • Which peaks should I use from the bioanalyzer for TruSeq Targeted RNA Expression?
    Perform a region analysis on the bioanalyzer and define the region from 150–250 bases to get the molarity for the entire sample.
  • How can I share my Nextera Rapid Capture Custom Enrichment design content with my collaborator?
    Custom target designs can be shared by saving Target Regions from your DesignStudio project to a .csv file using the Export function. The exported .csv file can be sent to your collaborator and imported into a new DesignStudio project, using the file upload method, to view the Target Regions of interest. Probe sequences can also be shared using the Export function of the Probes table.
  • Does Illumina recommend filters or cutoffs to use for different scenarios?

    No. Filtering parameters vary depending on the application and objective of the investigator.

  • How do I access MiSeq Reporter?
    MiSeq Reporter software runs as a web server that runs continuously on the MiSeq computer. To view current MiSeq Reporter status, open your web browser and enter the following URL: http://PCname:8042 (where “PC Name” is the name or IP address of the MiSeq PC).
  • What sequencing read length is supported on the HiSeq X system?

    Supported read lengths for the HiSeq X are up to 2 x 150 bp plus single 8 bp indexing. Only single-indexed runs are possible on the HiSeq X system.

  • What is the recommended insert size for sequencing on the HiSeq X system?

    The supported insert size on the HiSeq X system is 350 bp and 450 bp for TruSeq Nano DNA and TruSeq DNA PCR-Free libraries.

  • What are some third party data analysis tools for TruSeq ChIP samples?

    There are a number of third party solutions available for the analysis of ChIP data.  These include, but are not limited to, MACS, Avadis, and Partek.  Please note that Illumina cannot provide support for the use of third party software; please contact the software resources directly with any questions regarding the analysis and use of their software.  Additional literature references can be found on the Illumina Epigenetics webpage.

  • What is the length of the oligo baits used in Nextera Rapid Capture Enrichment reactions?

    The coding Exome (45 Mb) and Custom content assays use 80 mer oligos, while the Expanded Exome (62 Mb) uses 95 mer oligos.

  • Should TruSeq small RNA libraries be run as single-read or paired-end?

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

  • What are the workflow changes for analysis/demultiplexing on the MiSeq, HiSeq, and GA for dual-indexed libraries compared to single-indexed libraries?

    There are no changes for MiSeq analysis. HiSeq and GA data require an upgrade to CASAVA 1.8.2 to demultiplex dual-indexed libraries. It is also recommended to upgrade to SAV 1.8.4 or higher to use the new Index tab for real time demultiplexing information.

  • What are the workflow changes for analysis/demultiplexing on the MiSeq, HiSeq, and GA for dual-indexed libraries compared to single-indexed libraries?

    There are no changes for MiSeq analysis. HiSeq and GA data require an upgrade to CASAVA 1.8.2 to demultiplex dual-indexed libraries. It is also recommended to upgrade to SAV 1.8.4 or higher to use the new Index tab for real time demultiplexing information.

  • How does ForenSeq DNA Signature Prep work?

    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.

  • How can I get GenomeStudio software?

    The software is provided without charge and can be downloaded from the Downloads page. 

  • Can genotyping projects that were opened and modified in the PC Module be imported back into the GenomeStudio Genotyping Module?

    No, a polyploid project (.pcm) can only be opened using the PC Module.

  • Does the RNA Fusion Module provide information about gene expression or variant calls?

    The RNA Fusion Module does not provide variant call data. The RNA Fusion Module provides read counts in the samples/GeneCountsfolder. For detailed information on variants and gene expression, the BaseSpace RNA-Seq Alignment App is recommended.

  • How do I check the quality of my library?
    Illumina recommends the BioAnalyzer. Please see the Nextera DNA Sample Preparation Guide for more specific details and example traces.
  • Can I monitor my HiSeq run remotely?

    Illumina provides the Sequencing Analysis Viewer (SAV) software that can be run on a Windows PC to remotely monitor your run. The software does not allow any control over the run and requires that the PC is connected to the analysis server over the network. Another application you can use to monitor your run is SeqMonitor, which allows you to monitor your run using your iPhone or iPad.

  • Can I get variant information (e.g., SNP, indel) from the TruSeq Targeted RNA Expression sequence data?
    This information can be found in the .bam files. However, MiSeq Reporter will not perform variant calling or generate .vcf files. cSNPs can be analyzed and visualized using the Illumina Genome Viewer.
  • What are the main components in NuPCR reaction?
    NuPCR PCR reactions have just 3 components. Dilute NuPCR Master Mix, cDNA target, and NuPCR Assay reagents and you're ready to go.
  • How many amplicons can be processed at one time?

    Currently, this kit 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).

  • Can libraries prepared with this kit be combined with other library types in the same lane?

    Illumina does not support running libraries prepared by different sample prep kits in the same lane of a flow cell and does recommend running any combination of library types in a single lane. Illumina PhiX can be spiked in to any lane with user-prepared libraries.

  • Is a sample sheet/library sheet optional or mandatory for sequencing runs and analysis?

    For runs on the HiSeq, HiScanSQ, or GAIIx, creating and loading a sample sheet at the start of the run is optional. However, using a sample sheet allows you to view data shown on the indexing tab in the Sequencing Analysis Viewer (SAV) during the run. If you do not load a sample sheet at the start of a run in HCS, you will not be able to view indexing data in SAV. When analyzing indexed samples using CASAVA v1.8.2, a sample sheet is required. MiSeq runs require a sample sheet when setting up the run in MCS.

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

  • How do I know if the TruSeq controls have worked?
    • The TruSeq controls were not designed to be a qualitative metric of the efficiency of the various steps in the sample prep but rather an indicator of whether or not the step worked or not. Actual counts of the controls will vary based on sample type, input, etc.
    • In SAV (Sequencing Analysis Viewer), if you see counts for a particular control, this mean that this step has worked whereas if you see no counts (dark blue color), this step has likely failed. If the band has been cut across multiple insert sizes, you may see counts at different size increments.
  • How do I add more data (samples or lanes) to my GenomeStudio project?
    Create a new GenomeStudio project with all of the samples and lanes you want to include.
  • At what steps in the workflow can you multiplex samples?

    Samples are multiplexed before enrichment.  Samples can also be multiplexed before sequencing if loading multiple samples on a single flow cell.

  • What applications are suitable for Nextera?
    Nextera library preparation is suitable for genomic DNA applications where speed is paramount, input material is limiting (e.g., less than 100 ng DNA), where the sample prep throughput is a bottleneck, or when non-mechanical fragmentation is required. For example, Nextera is ideally suited to small genome sequencing with MiSeq.
  • How do I normalize between samples for TruSeq Targeted RNA Expression?
    In order to normalize your data, identify a gene from RNA-Seq or Microarray data that is constant across the samples being tested. Include assays that target this gene in your TOP panel when using DesignStudio. Additionally, this gene needs to be identified in the sample sheet, in a column with the header “Normalize”, so MiSeq Reporter will use it to normalize the data.
  • When generating a sample report, can I import sample information instead of entering it manually into VariantStudio?

    Yes. When you generate a report from the Sample Report menu, you can either type in the sample information requested in the chosen report template or import the sample information from an external text file.

  • What are the differences between “ChIP grade” and “non-ChIP grade” antibodies?

    Although we strongly recommend using antibodies that have been shown to produce good results in ChIP-chip experiments, there are exceptions. ChIP grade antibodies were designed specifically for ChIP, but many also work well in other applications and assays. Many antibodies have been successfully tested in ChIP, so please consult directly with the antibody provider for more information. See more info.


  • How do I quantify the final TruSeq Nano DNA libraries?
    qPCR or fluorometric quantification assays using dsDNA binding dyes (such as Qubit or Picogreen) can be used as methods of final library quantification.
  • Can I sequence TruSeq Custom Enrichment libraries on the MiSeq?

    Yes, you can sequence your enriched libraries on the MiSeq System, obtaining results in hours instead of days. However, the MiSeq Reporter currently cannot process the manifest file provided for your enrichment experiment. Therefore, the MiSeq Reporter maps your reads to the entire genome, not just the enriched regions, and alignment of the reads takes at least four hours.

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

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

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

  • Are the TruSeq Stranded RNA HT adapter plates compatible with the TruSeq RNA Sample Prep kit v1 or v2?

    The adapter plate is not compatible with TruSeq RNA v1 or v2 kits as the protocol and the reagents are different.

  • What methods of shearing are supported?

    The protocol is optimized for shearing using Covaris fragmentation. Other methods are not supported or tested by Illumina and can result in low yield, unexpected size distributions, or sample failure.

  • Do I need to log on to my BaseSpace Sequence Hub account to access BaseSpace Variant Interpreter?

    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.

  • Is there a minimum number of samples that must be processed together for ForenSeq DNA Signature Prep?

    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.

  • What tools are offered for data analysis?

    The following software tools are available for this kit: Illumina Experiment Manager, MiSeq Reporter, and the Illumina Amplicon Viewer.

    MiSeq Reporter is a simple, on-instrument software that demultiplexes indices/samples, performs alignment of reads, and creates a variant report immediately upon completion of each sequencing run. MiSeq Reporter also creates an assembly for each amplicon (on a per sample basis), per SNP calling, and generates graphs and reports including the sequencing coverage per amplicon.

    Illumina Amplicon Viewer performs data visualization from multiple runs and custom report generation off of the instrument. Analysis workflows are available for detecting both germline and somatic variation in sequenced samples.

  • Which part of the flow cell stage do I clean before loading a flow cell?

    Clean the entire flow cell stage within the flow cell compartment, including the glass surface of the optical alignment target, to remove any visible particulate. The flow cell stage consists of the two flow cell holders and surrounding area.

    Use an alcohol wipe for cleaning, and a lint-free tissue for drying and removing alcohol residue. Cleaning is necessary only if particulate is visible.

     

  • What are the DNA input requirements for ForenSeq DNA Signature Prep?

    Follow these DNA input recommendations:

    • 1 ng gDNA
    • 2 µl crude lysate
    • 1.2 mm FTA card punch per samples is required.
  • Is ethidium bromide used in the gel purification step of mRNA-Seq library preparation?
    Yes. You can add ethidium bromide or another DNA-visualizing fluorophore to the gel.
  • What is a recommended control sample for this protocol?

    Illumina recommends using Agilent Technologies Human UHR total RNA (catalog # 740000) as a control sample for this protocol.

    UHR contains the following known fusions: BCR-ABL1, BCAS3-BCAS4, and NUP214-XKR3 (at low levels).

  • What are the reference databases and links to support information regarding these resources?

    The Illumina VariantStudio software uses multiple annotation sources. For more information, see the VariantStudio User Guide.

  • What is the recommended amount of RNA library for enrichment?

    Use 200ng of each RNA library. All enrichments reactions with this kit are single-plex only. If you get lower than 200ng library yield, you can input the entire amount into the enrichment. However, sequencing results will vary in quality.

  • How do I start an array scan following a completed sequencing run on the NextSeq 550 system?

    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.

  • Is there any reason why I would rather not have stranded information?

    Projects that are in-progress may keep their current configuration and workflow rather than changing protocols in the middle of the study. Additionally, pooling reads from paired-end stranded runs is equivalent to the same number of reads from non-stranded single read runs (i.e., 2 million reads each of read 1 and read 2 pooled together (4M total) is identical to 4 million reads of read 1 of a non-directional library).

  • Which systems are capable of running HiSeq Rapid v2 chemistry?

    Any Hiseq system that has Rapid Run mode can run HiSeq Rapid Run v2 chemistry.

  • Can I change the design strand for individual targets or probes in Nextera Rapid Capture Custom Enrichment?
    No. DesignStudio uses only the forward strand for designing Nextera Rapid Capture Custom Enrichment assays for optimal performance across the probe panel.
  • Which magnetic stands are required for this protocol?

    This protocol requires 2 magnetic stands. For pre-PCR, Illumina recommends the DynaMag-96 Side Skirted Magnet (use with 96-well full-skirted PCR plates) or the DynaMag-96 Side Magnet (use with Eppendorf 96-well twin.tec PCR plates). For Post-PCR, Illumina recommends the Magnetic stand-96 (use with midi 96-well storage plates).

  • How is scanning enabled on the NextSeq 500 system?

    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.

  • I get the following error message, what should I do? ERROR: reference sequence /data/runs/genomes_human/c1.fa does not exists

     If you run into the ERROR: reference sequence ⁄data⁄runs⁄genomes_human⁄c1.fa does not exist, there is a good chance that you are not using files provided by Illumina.

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

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

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

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

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

  • What read lengths are recommended for each TruSeq Nano DNA Library Prep insert size?

    The recommended read length for the 350 bp insert size is ≤ 2 X 101 bp and ≤ 2 X 151 bp for the 550 bp insert size. Read lengths greater than those recommended for each insert size will produce a significantly higher percentage of overlapping read-pairs.

  • What are the differences between the ChIP-Seq sample prep kit and the standard genomic DNA sample prep kit?
    Sample Preparation Quality DNA that has been chromatin immunoprecipitated with chemical modifications Standard genomic DNA with no modifications
    Starting material 10ng 5μg
    Enzymes / adaptors Less is used More is used
    Product visible on gel No Yes
    Polymerase Standalone Part of pre-mixed component
    Quantitation of final product Requires sensitive method like a BioAnalyzer Less sensitive method required
  • What control software version is compatible with current kits and library prep methods?

    Use HiSeq X Control Software v3.1, or later, with the HiSeq X Reagent Kit v2 or HiSeq X Reagent Kit v2.5. Version 3.1 is required to sequencing TruSeq DNA PCR-Free libraries.

    Library Type

    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

  • Will the new Nextera dual-indexing support single-indexing as well?
    Yes. 12-plex single-index runs are supported on the HiSeq/GA using i7 indexes only. GA support for Nextera will be available in November 2011 when the latest version of SCS/RTA is available. Please see the Nextera DNA Sample Preparation Guide for more information on pooling recommendations for samples less than the full kit size.
  • How many reads do I need per sample?

    ~3 million reads per sample is recommended. More or less reads may be required depending on the expression level of the fusion.

  • How long are the amplicons?

    120-170 bp

  • Can I use index adapters that are not provided with NeoPrep library prep kits?

    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.

  • How long does it take to generate ready-to-sequence TruSeq Rapid Exome libraries?

    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.

  • To what level have you pooled BACs successfully?
    There are no inherent limits in the software. Illumina scientists have pooled 29 BACs of 130 kb each.
  • Is the sample sheet/library sheet optional or mandatory?
    For MiSeq runs, a sample sheet is required at the start of the run to enable analysis. For HiSeq/HiScanSQ/GA runs, creating and loading this sample sheet at the start of the run is optional, but is highly recommended in order to view data in the new indexing tab of SAV during the run. If you do not load a sample sheet at the start of a run in HCS, you will not be able to view indexing data in SAV. Additionally it is recommended to create the sample sheet in the Illumina Experiment Manager (IEM) prior to performing sample prep in order to confirm appropriate index combinations.
  • Can I use HiSeq X reagents on my other HiSeq system?

    No. HiSeq X reagents are designed and optimized for HiSeq X systems. HiSeq X reagents are not compatible with other HiSeq models.

  • Can I save intensities from a run on the HiSeq or HiScanSQ?
    Yes. The *.cif files can be saved and transferred to the analysis server over the network.
  • How do I obtain the Decode files required to scan a BeadChip on the NextSeq 550 system?

    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.

  • Can the PC Module call genotypes?

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

  • Are there changes to reagents in the MiSeq v3 kits?

    Yes. There are two new reagents in MiSeq v3 kits: new Incorporation Mix (IMT) and new Scan Mix (USM), and the PR2 volume is increased to 500 ml to support longer runs.

  • What percent of sequencing reads is expected to be on target?

    For good quality samples, > 90% of sequencing reads on target is expected for control samples.

  • Do I need a Paired-End Module (PEM) with the HiSeq?
    No. The HiSeq contains an integrated paired-end fluidics system and a reagent rack for paired-end reagents.
  • What DNA strand is used to design my targets in DesignStudio for Nextera Rapid Capture Custom Enrichment?
    The forward strand is used.
  • Can I expect to see any changes in intensity with HCS v1.4 or HCS v1.5?

    Intensity for the G channel is expected to be lower, but the rate of decay is much slower due to one of the new reagents in the TruSeq SBS Kit v3 - HS called SRE. Therefore, your data quality will not be impacted.

  • What is the difference betweeen the TruSeq Nano DNA Library Prep LS and HS protocols?

    The TruSeq Nano DNA Library Prep Guide contains both a Low Sample (LS) and High Sample (HS) protocol. These protocols differ in the types of plates used and the method of incubation and mixing. The LS protocol uses 0.3 ml PCR plates, the incubation steps are done on a thermal cycler, and mixing method is pipetting. The HS protocol is done in MIDI plates, requires additional equipment such as microheating system for all incubation steps, and mixing method is done on a microplate shaker. The LS protocol is optimized for processing 24 samples at a time and HS protocol for more than 24 samples. Both the LT and HT kits can be used with either the LS or HS protocol and will give comparable results; however, the HS protocol can yield more consistent results between samples.

  • How much genomic content can I target in my Nextera Rapid Capture Custom Enrichment project?
    Nextera Rapid Capture Custom Enrichment is available for designs that include 2000–67,000 total probes. Depending on probe density within the designed regions, this represents about 0.5–15 Mb of genomic content.
  • Are my existing Illumina sequencing libraries compatible with the HiSeq?
    Yes. Single read and paired end libraries for RNA and DNA applications are compatible with the HiSeq.
  • What is a patterned flow cell?

    A patterned flow cell is a flow cell with billions of ordered nano-wells that are manufactured into the flow cell glass. The ordered wells allow for the generation of sequencing clusters in an ordered arrangement. Clusters are aligned more closely together increasing the number of output reads and amount of sequence data generated. The HiSeq 3000/4000 patterned flow cell contains 8 lanes and has the same general dimensions as a HiSeq high-output flow cell.

  • What is the typical library size distribution for my final TruSeq Synthetic Long-Read DNA libraries? What is the expected yield?
    Expected final pooled libraries range from 200–3000 bp when assessed on a Agilent Technologies 2100 Bioanalyzer using a High Sensitivity DNA chip. Illumina has observed a wide range of concentrations (20-200 nM) depending input gDNA quality, operator technique, and other variables.
  • Is the incorporation reagent supposed to be a different color?

    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.

  • Can I run differential expression analysis on my data?

    Yes, use the Cufflinks Assembly & DE BaseSpace App.

  • How long after completing a sequencing run can I start an array scan on the NextSeq 550 system?

    You can start an array scan when the NextSeq 550 system completes the automatic post-run wash following a sequencing run.

  • Why does the Nextera Rapid Capture Enrichment protocol require the TruTemp Heating System and MIDI plate insert?
    The MIDI heat block used for tagmentation better supports automation and reduces the number of plate-to-plate sample transfers. The MIDI plate insert fits the 96-well, 0.8 ml "MIDI" plate used in the protocol.
  • Are the Illumina adapter sequences available?

    Illumina adapter sequences are provided in the Illumina Customer Sequence Letter. This kit uses the TruSeq adapters.

  • What is the minimum amount of samples that I have to prepare using the TruSeq Exome protocol?

    One sample can be prepared at a time. However, the enrichment reagents are the limiting factor. Illumina recommends a minimum of 3 samples for this kit.  It is most economical to process and pool for enrichment the maximum number of samples supported for the kit configuration.

  • Should I leave my MiSeq on when not in use?
    It is best to leave the instrument on at all times. However, if you need to turn off the instrument, perform a maintenance wash first, and then use the Shut Down command on the Manage Instrument screen to safely shut down Windows. If the instrument will not be used within seven days, make sure that you prepare the fluidics system to sit idle. For more information, see the MiSeq System User Guide, Part # 15027617.
  • Are libraries prepared with TruSeq Stranded RNA HT sample prep kits compatible with all Illumina sequencers?

    Yes, these libraries can be used on all HiSeq, MiSeq, HiScanSQ and GA instruments.

  • Are libraries prepared with TruSeq Stranded RNA HT sample prep kits compatible with all Illumina sequencers?

    Yes, these libraries can be used on all HiSeq, MiSeq, HiScanSQ and GA instruments.

  • How is the instrument health upload option communicated to customers?

    The first time the HCS 2.2 is launched, you will see a notification regarding instrument health data. This notification 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.

  • Can I use third-party analysis options with MiniSeq sequencing data?

    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.

  • Can I analyze .cif files with BaseSpace?

    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.

  • Do I have to remove the blue dye from a NeoPrep library before sequencing?

    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.

  • How many samples can be processed per TruSeq Sample Prep v1, v2 or LT Kit?

    TruSeq DNA and RNA Sample Prep kits - Set A

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

    TruSeq DNA and RNA Sample Prep kits - Set B

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

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

  • What data quality can I expect from a run on the HiSeq X system?

    You can expect greater than 75% of bases above Q30 with a 2 x 150 bp run using the following libraries:

    • A human library prepared with the TruSeq Nano DNA Sample Prep Kit (350 bp or 450 bp insert)
    • A human library prepared with the TruSeq DNA PCR-Free Sample Prep Kit (350 bp or 450 bp insert)
    • Illumina PhiX control library
  • How many different species can the MiSeq metagenomics workflow identify?
    The MiSeq metagenomics workflow identifies genera, not species, and can identify 1311 different genera.
  • What does the data look like? Is there demo data available?

    For an example of data from an experiment, see the data set available on BaseSpace. The example data set is from the TruSeq Amplicon Cancer Panel sequenced with miSeq on FFPE-derived DNA.

  • How many samples can I process at the same time with the ForenSeq DNA Signature Prep Kit?

    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.

  • Are FASTQ files generated in addition to BCL files?
    BCL files are generated by RTA and FASTQ files are generated in post run analysis by MiSeq Reporter.
  • Does Illumina provide antibodies for ChIP assays?

    No, Illumina does not provide antibodies.


  • Which applications are supported on the MiSeq?

    MiSeq Reporter, the secondary analysis software on the MiSeq, supports amplicon sequencing, targeted resequencing, small genome sequencing (de novo/resequencing), and small RNA applications. For complete information, see Sample Prep Applications for the MiSeq System.

  • What sequencing read length is supported on the HiSeq 4000?

    Supported read lengths for the HiSeq 4000 are up to 2 x 150 bp. Three kit sizes (50-cycle, 150-cycle, and 300-cycle) are available to support a range of read lengths.

  • Are there considerations for combining certain indexes or only combining 1-2 indexes?

    Testing has shown that performance shows minimal variability when combining low numbers of indexes, or across particular combinations of indexes, so any number or combination may be used.

  • What are the requirements for importing whole genome data or large gVCF files?

    Importing large gVCF files requires more memory and takes longer to import. Use the following information as a guideline to estimate required RAM. This information is based on a Quad-Core Xeon® processor with 16 GB RAM.

    • Exome: 500-800 MB RAM per annotated exome
    • Whole genome: 3.5 GB RAM per annotated whole genome, 4 GB RAM recommended in addition to system memory. For example, if you are importing three annotated whole genome files, you need at least 12 GB but 16 GB is recommended to account for system memory.
    • Whole genome gVCF without hom-ref positions: 4.0 GB RAM per annotated whole genome in addition to system memory. VariantStudio imports non-hom-ref positions but it takes longer than 6 hours to go through all 300-400 million lines in the gVCF file. If you are not using the hom-ref import option, Illumina recommends using standard VCF files.
    • Whole genome gVCF with hom-ref positions: VariantStudio populates a maximum of 10 million rows in the Variants table. Therefore, it is not possible to import all positions. When all 10 million lines are populated, > 5 GB RAM without annotation is required. With annotation, 6-7 GB RAM is required. Alternatively, you can pre-process gVCF files for regions of interest before importing to VariantStudio.
  • What is the overlap in targeted genomic content between the Nextera Rapid Capture Exome and Expanded Exome Enrichment products?

    89% mega bases targeted in the Nextera Rapid Capture Exome Enrichment overlap with the 62.1 mega bases of regions targeted in the Expanded Exome Enrichment pool. BED files outlining both the overlapping regions as well as the content unique to each product can be downloaded from the Nextera Rapid Capture Exome and Expanded Exome Enrichment Kits Downloads support page.

  • Are there any additional components I need to order to complete the prep?
    In addition to either the Nextera XT DNA Sample Preparation Kit for 24 Samples (catalog# FC-131-1024) or 96 samples (catalog# FC-131-1096), there are corresponding Nextera XT Index Kits for 24 Indices/96 samples (catalog# FC-131-1001) and 96 Indices/384 samples (catalog# FC-131-1002). Please note that these indices are optimized for the Nextera XT kit and are not interchangeable with the Nextera kit.
  • Can TruSeq Synthetic Long-Read DNA libraries be combined with other library types on the same flow cell?
    Yes, a sample sheet is used to specify how each lane is to be analyzed. However, it is critical that TruSeq Synthetic Long-Read DNA library sequencing parameters (2 x 101 + 8 bp index) apply to the entire flow cell. If multiple TruSeq Synthetic Long-Read DNA libraries are run on the same flow cell, each must be run on a separate lane, because the same barcoding scheme is utilized for each sample.
  • What is the estimated overall time for preparing libraries with the Nextera XT kit?
    The estimated time for 8 samples is approximately 90 minutes for the prep and 70 minutes for the bead based normalization (BBN). The estimated time for 96 samples is approximately 3 hours for the prep and 90 minutes for the BBN.
  • Can I use HiSeq X patterned flow cells on other platforms?

    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.

  • What will happen if I use less than 1 µg of total RNA?
    The overall yield and quality of the library can diminish with lower input amounts. The PCR cycles in the enrichment step of the protocol (following the gel) are tuned for a certain amount of input material. The protocol calls for exactly 15 cycles of PCR amplification. If the amount of material purified from the gel is significantly less than the standard 1–10 µg prep, then you might need more PCR cycles to create a clearly visible band. However, we do not recommend over amplifying the library, since this can lead to artifacts during the electrophoresis and quantification of the library.
  • Do TruSeq Stranded Total Stranded Sample Prep Ribo-Zero kits only remove ribosomal RNA?

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

  • Does the MiSeq have an instrument PC?

    Yes. The MiSeq includes a fully-integrated instrument computer that runs on Windows 7 Embedded and contains the operating software, control software, and analysis software.

  • Are TruSeq HT sample prep index sequences for the first 7 cycles of the first index read the same as TruSeq v2/LT kits, TruSeq Custom Amplicon kits, Illumina Nextera, etc.?
    No, the TruSeq HT adapter plates contain 12 new and unique sequences that are used for the first index read. For specific index sequences, please refer to the Illumina Customer Sequence Letter, the Illumina Experiment Manager (IEM), or the appropriate sample preparation guide. The sequencing primer sequences are proprietary and not available.
  • How large is the run folder?
    Saving the standard set of files without thumbnails results in a run folder that is approximately 200–500 GB. If standard thumbnails are saved, approximately an additional 300 GB is required. Approximately 1 TB is storage used by the alignment folder and related folders.
  • What is the difference between TruSeq Stranded Total RNA Sample Prep with Ribo-Zero Human/Mouse/Rat and TruSeq Stranded Total RNA Sample Prep with Ribo-Zero Gold?

    The Ribo-Zero Human/Mouse/Rat kit depletes samples of cytoplasmic ribosomal RNA. The Ribo-Zero Gold kit depletes samples of both cytoplasmic and mitochondrial ribosomal RNA.

  • Can I use input RNA from FFPE tissues for TruSeq Targeted RNA Expression?
    Yes, Illumina recommends using 100-200 ng degraded or FFPE samples with an average fragment size of ≥ 200 bp for optimal hybridization. In addition, check the total RNA integrity following isolation on an Agilent Technologies 2100 Bioanalyzer using an RNA 6000 Nano Chip. For more information, see the RNA Input Recommendations section of the TruSeq Targeted RNA Expression Guide.
  • What data compression options are available for high output runs using HCS v2.2?

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

  • Do I need a cBot instrument to run sequencing v4 flow cells on my HiSeq?

    Yes, a cBot is needed for cluster generation on any 8-lane high output flow cell, including HiSeq v4 and TruSeq v3.

  • How is strandedness maintained after DNA amplification?
    Strandedness is maintained via the directionality of the adapters. The p7 adapter will be on the 3' end of the cDNA strand. As a consequence, the cDNA strand is sequenced. Second strand synthesis is performed using dUTP in place of dUTP and a high fidelity polymerase that cannot process through dUTP template is used for PCR enrichment. As a result, only the first strand product is amplified and the strand information is retained based on the p5 and p7 adapter orientation.
  • How long does it take to perform a post-run wash on the MiSeq?

    The post-run wash takes approximately 30 minutes.

  • What is the shelf-life of TruSeq Synthetic Long-Read DNA library prep kits?
    Expiration dates are printed on the kit box labels and reagent tubes. Illumina guarantees 3 months of shelf life from the time of kit shipment.
  • Does MiSeq Reporter recognize *.fasta or *.fa?
    Yes, MiSeq Reporter recognizes both extensions.
  • What does it mean if you have a fusion detected but there are 0 reference reads for Gene 1 or 2?

    Reference reads refers to reads/read-pairs that support structurally normal genes at that fusion breakpoint. The 0 means that there is no evidence of structurally normal genes in the RNA-Seq data; this is distinct from “whole gene counts” which is shown in the last table of the report. 

    Additional rearrangements and duplications that occur in concert with the fusion could also affect this number. 

  • Do the TruSeq Stranded Total RNA Sample Prep Kits with Ribo-Zero Globin or Plant work on degraded RNA?
    All Ribo-Zero kits are compatible with degraded RNA. For more information, see the TruSeq Stranded Total RNA Sample Preparation Guide.
  • How much mixing is required?

    Follow the protocol as described in the reference guide for your kit. The procedures require frequenent and complete mixing for successful rRNA removal.

  • What indexing options are available on the HiSeq 4000?

    The HiSeq 3000/4000 reagent kits support single and dual indexing options. SBS kits includes sufficient reagent for 25 additional cycles for indexed sequencing. The indexing primer provides primers for both single and dual indexing.

  • Does TruSeq RNA Sample Prep provide information about the originating strand from which the DNA was transcribed?

    In the random priming process to generate cDNA with the best coverage, this information is not retained. If originating strand information is required, please refer to the Directional mRNA-Seq Sample Preparation Guide or other published applications.

  • Is there a rehyb protocol available for HiSeq v4 runs?
    Yes. Primer rehybridization for HiSeq v4 runs can rehyb the Read 1 primer, the Index 1 Read primer, or the Read 2 primer. Rehyb runs are performed on the HiSeq. For more information, see the HiSeq Primer Rehybridization Reference Guide (part # 15050105).
  • What export formats are available from Illumina VariantStudio?

    In VariantStudio, you can export information displayed in the Variants table, which includes variants and associated annotations, in a TSV file format. Sample reports are generated in either RTF or PDF file formats.

  • How is the default value of 100,000 determined for minimum breakpoint distance?

    The default value was determined empirically from our test dataset. This was a safeguard to prevent calling of really obvious read-through transcripts from being called as fusions. 

  • Where are the content manifest file downloads for my TruSeq Targetetd RNA Expression assay pool?
    Manifest files can be downloaded using the Export Manifest function in Project Dashboard of your DesignStudio project. The manifest file becomes available once your order ships, not when the order is placed or designed in DesignStudio.
  • How many samples can be processed per TruSeq Nano DNA LT and HT Library Prep Kit?

    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.

  • Does VariantStudio perform variant classification?

    No, you perform variant classification. With VariantStudio, you can provide annotations that can be used to classify variants within VariantStudio. After you assign a classification category to a variant, the information is saved to the Classification Database so that it can be applied to the same variant observed in other samples.  Classifications saved in the Classification Database can be applied automatically to other samples through the Apply Classifications from Database menu.

  • How many indices are available for the TruSeq ChIP Sample Prep Kit?

    There are a total of 24 unique indices: 12 in Set A and 12 in Set B.

  • Is the BaseSpace Variant Interpreter software validated?

    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.

  • The flow cell is loose in the cartridge, do I need a replacement?

    No. The flow cell is designed to be loose in the cartridge to ensure proper registration.

  • How do I run my data through the RNA-Seq Alignment App?

    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.

  • What is the library size distribution for TruSeq ChIP Sample Prep libraries?

    The final library should show a size distribution from ~250–300 bp.

  • How many cycles of sequencing do small RNA libraries need?

    This depends on the aim of the experiment. For expression profiling projects, we do not recommend longer than 36 cycles of sequencing. The small RNA molecules are normally 15–30 bases long, and sequencing beyond this point only sequences the adapter. Eighteen cycles may be sufficient. For discovery projects, it may be worthwhile to do 40–50 cycles. This will sequence into the adapter, but the adapter sequence will be long enough to be unambiguously identified and removed even on longer small RNA molecules.

  • How many cycles of sequencing do small RNA libraries need?

    This depends on the aim of the experiment. For expression profiling projects, we do not recommend longer than 36 cycles of sequencing. The small RNA molecules are normally 15–30 bases long, and sequencing beyond this point only sequences the adapter. Eighteen cycles may be sufficient. For discovery projects, it may be worthwhile to do 40–50 cycles. This will sequence into the adapter, but the adapter sequence will be long enough to be unambiguously identified and removed even on longer small RNA molecules.

  • Can I use Sequencing Analysis Viewer (SAV) to view data from a HiSeq 4000 run?

    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.

  • Do TruSeq HT dual-indexed libraries still sequence 1 bp more than the index sequence used for demultiplexing?

    No. Dual-indexed libraries sequence eight bases of actual index sequence rather than six bases plus one more for phasing calculations. For short sequences there are no phasing/prephasing corrections, so it is not necessary to sequence one additional cycle.

  • Is the GenomeStudio ChIP-Seq Module compatible with CASAVA 1.8?

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

  • How do I visualize my Nextera Rapid Capture Enrichment designed targets in a genome browser?

    Use the DesignStudio table Export function to save designed regions, targets, and probes in *.bed file format. Exported *.bed files can be imported into a genome browser, such as the Broad Institute Integrative Genome Viewer (IGV), which can be downloaded and run on your local PC computer, or the online UCSC Genome Browser.

    Note:

    • The UCSC Genome Browser may not accept very large *.bed files with many annotations.
    • Illumina recommends adding the track name annotation to your *.bed files when viewing multiple custom tracks, such as probes and gaps, in the UCSC Genome Browser. The *.bed files exported from DesignStudio do not contain a header with a defined track name. Therefore, when loaded into the UCSC Genome Browser, they appear with the generic track name of 'User Track' and description of 'User Supplied Track' and will overwrite any other previously uploaded unnamed tracks.
  • Can I create my own manifest file to analyze my data?

    No. 

  • How can I combine two regions in close proximity into one larger region?
    When you create a merged table, you can specify how close two regions must be in number of base pairs.
  • Why do we need to align to the RNA Fusion Reference Genome?

    Calling novel fusion partners requires whole genome alignment. The RNA Fusion Module aligns reads to the Minimal Reference Genome using Spliced Transcripts Alignment to a Reference (STAR).

  • Can I sequence Nextera Rapid Capture Enrichment and TruSeq Rapid Exome libraries on the same flow cell as other Nextera libraries?

    Yes. All Nextera-based libraries use the same sequencing and index primers (HP10, HP11, HP12) and can be loaded in different lanes of the same flow cell for sequencing.

  • How many PCR cycles are required in the preparation of the TruSight Myeloid libraries?
    The TruSight Myeloid library preparation requires 27 cycles of PCR.
  • What is the difference between Call Rate and Poly Call Rate in the Samples Table of the PC Module?

    Call Rate is carried over from the Samples Table in the GenomeStudio Genotyping Module in which the original genotyping project (.bsc) was created. Entries in the Call Rate column do not change when SNPs are clustered in the PC Module. In contrast, the Poly Call Rate is calculated from clustering SNPs in the PC Module and represents the percentage of SNPs for which a given sample was assigned to a cluster.

  • What cluster generation kits are compatible with TruSeq RNA Access libraries?
    For kit compatibility information, see the Illumina Version Compatibility Reference.
  • What types of controls should be run for a ChIP assay?

    Common controls include no primary antibody or use of normal rabbit IgG in ChIP as a negative control. In most cases, if you are studying the same factor in different tissues or cell lines, one control is sufficient. More controls will be needed if you are studying multiple factors from the same source.

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

  • Is a PhiX control lane necessary with small RNA libraries?
    Many small RNA libraries do not require a PhiX control lane to generate accurate matrix and phasing estimates. This is especially true in the case of samples from organisms with balanced genomes (e.g. mammalian organisms). Libraries from organisms with unbalanced genomes, or libraries with a high proportion of reads from a single template (for example, adapter reads, or samples from a tissue that expresses a single small RNA at high levels), may require use of a control lane for matrix and phasing generation. In any case, a PhiX control lane acts as a positive control for run performance and allows error rate estimation.
  • What are the required input amounts for TruSeq Nano DNA Library Prep?

    The protocol has been developed and validated with 100 ng of high quality DNA for a 350 bp insert size and 200 ng of high quality DNA for a 550 bp insert size.

  • Should the NeoPrep System be placed in a pre-PCR or post-PCR area?

    Illumina recommends locating the instrument in a post-PCR laboratory, because the final product collected from the instrument is amplified libraries.

  • What regions are included in the panel?

    The following table lists the genes and exons included in the panel.

    Gene

    Target or Region

    Potential Disease States

    AKT1

    Exon 3 (partial)

    Breast

    BRAF

    Exon 15 (partial)

    Melanoma, Colon, Lung

    EGFR

    Focal Amplification, Exon 12 (partial),
    18, 19, 20, 21 (partial)

    Lung

    ERBB2

    Focal Amplification, Exons 14 (partial),
    17, 18, 19, 20 (partial), 21 (partial), 24, 26

    Breast, Lung

    FOXL2

    Exon 1 (partial)

    Ovary

    GNA11

    Exon 5 (partial)

    Melanoma

    GNAQ

    Exon 5 (partial)

    Melanoma

    KIT

    Exons 8, 9, 10, 11, 13, 14, 17, 18

    Gastric, Melanoma

    KRAS

    Exon 2 (partial), 3 (partial), 4

    Colon, Gastric, Lung

    MET

    Focal Amplification

    Lung, Colon, Gastric

    NRAS

    Exon 2 (partial), 3 (partial), 4

    Colon

    PDGFRA

    Exon 12, 14, 18

    Gastric, Melanoma

    PIK3CA

    Exon 10, 21

    Lung, Breast, Prostate

    RET

    Exon 16

    Lung

    TP53

    Full CDS

    Lung, Melanoma, Ovary, Colon

  • Which organisms are supported for TruSeq Targeted RNA Expression?
    TruSeq Targeted RNA Expression is currently supported only for human, mouse, and rat.
  • What organisms do the TruSeq Stranded Total RNA Sample Prep kits support?

    The TruSeq Stranded Total RNA Sample Prep kits with Ribo-Zero Human/Mouse/Rat and TruSeq Stranded Total RNA Sample Prep kits with Ribo-Zero Gold both support human, mouse, and rat organisms. That use of these kits with other organisms has not been tested and is not supported.

  • What method should I use to quantify my input genomic DNA for TruSeq Synthetic Long-Read DNA library prep?
    Illumina recommends using fluorometric based methods for quantification, such as Qubit or PicoGreen, to provide accurate quantification for dsDNA. UV spectrophotometric based methods, such as the Nanodrop, measure any nucleotides present in the sample including RNA, dsDNA, ssDNA, and free nucleotides. This can give an inaccurate measurement of gDNA.
  • What are typical output concentrations from the pre-enriched Nextera Rapid Capture Enrichment library and the fully enriched final library?

    Typical output concentrations from the pre-enriched library are 40–125 ng/μl.

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

    • 1-plex: 5–50 nM (1.5–13 ng/μl)
    • 6-plex: 45–130 nM (12–34 ng/μl)
    • 12-plex: 100–250 nM (26–66 ng/μl)
  • What software is compatible with cBot 2?

    Use cBot software v3.0.40 or later. Previous versions of cBot software are not compatible.

  • Which species are available in DesignStudio for creating a Nextera Rapid Capture Custom Enrichment project?
    The human genome (hg19 build) is currently available for custom enrichment projects.
  • Can I use FFPE-derived total-RNA?
    We performed this protocol on FFPE-derived total RNA with good results. The poly-A purification step efficiently removes the ribosomal RNA in the sample, and the highly degraded transcripts that are present are purified by binding to the Poly-T beads. The cDNA fragments created from FFPE will be only as long as the length of mRNA that is still attached to the poly-A tail in each molecule. For example, if the RNA purified from an FFPE sample is degraded to an average length of 200 bps, then you will only get reads in the last 200 nts of most mRNA molecules in that sample.
  • What is the maximum amount of amplicons I can sequence on a MiSeq?

    If you are using the TruSeq Custom Amplicon Kit, 96 samples can be pooled in a single run combining up to 384 amplicons per sample.

  • Which parameters can be configured to manipulate the default clustering algorithms?

    Minimum Number of Points in a Cluster, Cluster Distance, and Maximum Number of Clusters in the SNP Table, which can be found in the Clustering Options dialog box.

  • How do I visualize my TruSight One targets in a genome browser?

    TruSight One regions, targets, and probes are provided in *.bed file format and can be found by visiting the TruSight One Product Support page.  The *.bed file can be imported into either the UCSC Genome Browser (genome.ucsc.edu) or the Integrated Genome Viewer (IGV) (www.broadinstitute.org/igv/).

  • Are the probes directed to a single strand?

    For the majority of target regions, a single strand is captured and sequencing data are highly stranded except in a subset of regions.

  • What genomes and databases are used for alignment and variant detection?
    The MiSeq comes with pre-installed databases, which include miRbase, dbSNP, and refGene. Also included are eight genomes: arabidopsis, cow, DH10b, hg19, mouse, rat, yeast, and s. aureus. You can upload your own references in fasta format (*.fasta or *.fa).
  • Which assays and platforms are compatible with the PC Module?

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

  • How do I know if I need to throttle BaseSpace, and how do I apply throttling?

    The BaseSpace Broker is designed to upload data to BaseSpace as soon as the data 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:

    1. In Windows, open a cmd window and open the Local Policy Editor. Run the program gpedit.msc.
    2. Expand the Computer Configuration / Windows Settings nodes.
    3. Select Policy-based QoS.
    4. Right-click Create new policy.
    5. Enter a name, such as Limit BaseSpace upload.
    6. Clear the Specify DSCP value.
    7. Select Specify Outbound Throttle Rate and enter 3 MBps (3 Mbytes/sec, or 24Mbit/sec), which is sufficient to allow data transfer to catch up.
    8. Click Next.
    9. Select Only applications with this executable name and enter Illumina.BaseSpace.Broker.exe.
    10. This policy applies to any source IP and target IP addresses. Click Next.
    11. This policy applies to all ports and protocols. Click Finish.
  • How long does it take to complete a run on the MiSeq?

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

  • What is the difference between the TruSeq sample prep HS and LS protocols?

    The TruSeq sample prep high sample (HS) protocol requires additional peripheral equipment, but reduces user touch-points and is ideally suited for projects that include more than 48 samples prepared at one time. The low sample (LS) protocol requires minimal peripheral equipment, but requires more user manipulation and is best suited for projects that include 48 or fewer samples. For specific differences in the workflows, including required equipment, refer to the user guide for the TruSeq sample prep kit that you are using.

  • Is it a problem to run low numbers of indices/pooled samples? What will happen if I run improper index combinations?

    If there is no signal in one of the color channels of the index read, the image registration might fail and no base will be called from that cycle. If no base is called, the index read may not be able to be matched to the sequence specified in the sample sheet, and then samples will not be able to be demultiplexed.

  • What kit sizes are available?

    The NovaSeq 5000/6000 S2 Reagent Kit is available in 3 sizes: 300 cycles (2 x 150), 200 cycles (2 x 100), and 100 cycles (2 x 50 or 1 x 100).

  • What tools are offered for data analysis with TruSight DNA Amplicon?
    The following software tools are available for TruSight DNA Amplicon: Illumina Experiment Manager, MiSeq Reporter, Illumina Amplicon Viewer, and VariantStudio. The MiSeq Reporter is simple, on-instrument software that demultiplexes indexes and samples, performs alignment of reads, and creates a variant report immediately upon completion of each sequencing run. The MiSeq Reporter also creates an assembly for each amplicon (on a per sample basis), performs SNP calling, and generates graphs and reports including the sequencing coverage per amplicon. The Illumina Amplicon Viewer performs data visualization from multiple runs and custom report generation off the instrument.

    The Illumina VariantStudio desktop application is available with purchase of TruSight panels and includes a one-year license to the VariantStudio desktop application. VariantStudio imports variant call files generated during analysis of sequencing data and provides commands to annotate variants. VariantStudio filters results using various filtering options, classifies variants according to their biological impact, and exports results to a report.
  • What is a BVP1?

    BVP1 is the Amplicon oligo tube, which is the final pool of oligonucleotide probes used to generate amplicons in the assay. A single BVP1 tube supports the amplification of all 265 amplicons in a single TruSeq Bovine Parentage reaction.

  • What is the workflow for dual indexing?

    See the appropriate HiSeq instrument user guide for details on the loading of reagents with different workflows and which primers you need to use for your library type.

    • Read 1: An indexed Read 1 follows the standard Read 1 protocol using reagents provided in the TruSeq SBS Kit. The Read 1 sequencing primer is annealed to the template strand during the cluster generation process on the cBot (HP6 or HP10).
    • Index Read 1 (i7): Following the completion of Read 1, the run proceeds to Index Read preparation. The Read 1 product is removed and the Index 1 (i7) sequencing primer (HP8 or HP12) is annealed to the same template strand. The run proceeds through 8 cycles of sequencing to read the Index 1 (i7).
    • Index Read 2 (i5): For paired-end flow cells, the Index 1 (i7) Read product is removed and the template anneals to the grafted P5 primer on the surface of the flow cell. The run proceeds through an additional 7 chemistry-only cycles (no images are taken) followed by 8 cycles of sequencing to read Index 2 (i5).
      For single-read flow cells, the Index 1 (i7) Read product is removed and the Index 2 (i5) sequencing primer (HP9) is annealed to the same template strand. The run proceeds through 8 cycles of sequencing to read the Index 2 (i5).
    • Paired End Resynthesis: Read 2 re-synthesis for dual-indexed paired-end sequencing uses reagents provided in the TruSeq PE Cluster Kit.
    • Read 2: Read 2 follows the standard paired-end sequencing protocol using standard SBS reagents.
  • What is the throughput of the HiSeq X system?

    Each system can generate 1.6–1.8 Tb in less than 3 days with greater than 75% of bases about Q30 from a 2 x 150 bp run. This throughput enables 16 genomes covered at 30x per run per system. For more information, see the HiSeq X system performance specifications page.

  • What is a high confidence fusion call?

    A high confidence fusion call means that a fusion is meeting the threshold filters, which are based on scores from calculated values of split read scores, paired read scores, break-end homology, and several other features. For more information about the calculations, see the Local Run Manager RNA Fusion Analysis Module Workflow Guide (document # 1000000010786).

  • Do we need to upgrade our servers for analysis of HiSeq data?
    You may need to upgrade your computing infrastructure to accommodate the new data output rate (Gigabases/hr) of the HiSeq 1500/2500 in Rapid Run mode, which is roughly twice the data output rate of a High Output run. However, Illumina offers two new methods to reduce the data output rate by more than 50% without affecting data quality, allowing you to leverage your existing infrastructure to store either twice the amount of data with the same amount of storage (High Output mode) or to keep the Rapid Run mode data output rate the same as your HiSeq 1000/2000 data output rate. The recommended configuration for one HiSeq is an IlluminaCompute Standard appliance. More information is available here or from your account manager.
  • How do I view my analysis results?

    In BaseSpace, click your Projects folder. Select Analysis and select the app session name that you saved your analysis under.

  • What is the maximum read length for a sequencing run?

    The NovaSeq Sequencing System supports read lengths up to 2 x 150 bp.

  • How do I order probes for TruSeq Targeted RNA Expression?
    Use DesignStudio to order probes. You will need a MyIllumina account.
  • How many samples are there per index pair in one TruSeq HT sample prep kit?

    Because each well of the 96-well HT adapter plate is single-use only, only one sample per index pair can be generated.

  • What version of bcl2fastq is supported for the NovaSeq System?

    bcl2fastq v2.19 is supported for processing NovaSeq data.

  • What consumables and equipment do I need to prepare TruSeq Targeted RNA Expression libraries in my lab?

    A full list of user supplied items can be found in the Consumables and Equipment section of the TruSeq Targeted RNA Expression Guide. Comparable performance is not guaranteed when using alternate equipment.

  • Can I use MiSeq Reporter software to analyze TruSight DNA Amplicon data from a NextSeq or HiSeq system?

    No, file directory structures are incompatible with MiSeq Reporter software. 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.

  • What method is recommended to quantify my tagmented library before sample pooling?

    Quantify the tagmented library using a fluorometric-based method specific for double-stranded DNA, such as QuantiFluor or PicoGreen. For more information, see the DNA Input Recommendations section of the library prep reference guide.

  • 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. Poor results may be obtained with low quality FFPE samples.

  • How long does the cBot take to perform the template hybridization procedure on a 2 lane flow cell?
    Just under one hour.
  • Do I need to prime SBS reagents in Rapid Run mode?

    No. Priming is integrated into the HCS workflow for Rapid Run mode.

  • How do I analyze the array data from the NextSeq 550 system?

    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.

  • What sequencing parameters are recommended for this kit?

    The recommended sequencing parameters is a 1 x 121 with dual index.

  • Can I switch modes on only 1 side of my HiSeq 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.

  • What does the Fusion Score mean?

    The score reflects the confidence in the fusion call, where 0 is low and 1 is high. Scores > 0.6 are reported as high confidence fusion calls.

  • Can I run Mate Pair libraries on the MiSeq?
    Yes. However, the MiSeq Reporter software does not have an analysis workflow for Mate Pair libraries. To perform de novo assembly, set MiSeq Reporter to generate FASTQ files, and then export them into a de novo assembler that will assemble Mate Pair libraries.
  • What if I don't have enough template DNA?
    The library process can be optimized to reduce the amount of DNA required. For some applications such as ChIP-Seq where DNA amounts might be low, steps such as size selection on a gel can be skipped. Instead, end-repaired DNA fragments can be ligated, purified by a column clean-up step that removes primer-dimers, and then quantitated and directly introduced to a flow cell for cluster amplification. For genomic DNA from small samples, fragmentation can be optimized to increase the number of DNA fragments that are within the target size range. If these DNA fragments are within a tight size range, then the agarose gel step can be eliminated.
  • Is there sample data I can view?

    Data sets are available on the BaseSpace Public Data page under the “Methyl-Seq” category.

  • Where can I get the adapter, primer, and index sequences used in the TruSeq sample prep kits?

    The adapter sequences are included in the Illumina Customer Sequence Letter. Additionally, the index sequences are available in the user guide for the TruSeq sample prep kit that you are using. Please note that the oligos are not sold separately. Primer sequences are proprietary.

  • Why does Illumina recommend paired-end sequencing of stranded RNA libraries?

    Due to the directional nature of the TruSeq Stranded RNA assays, paired-end sequencing captures the ends of the RNA molecule.

  • What quantitation methods does Illumina recommend for final library quantitation?
    Double Stranded DNA specific dyes, such as QuBit or picogreen.
  • Would there be issues demultiplexing current TruSeq v2/LT (single-index) libraries if a dual index run was done?

    No. You must specify the correct --use-bases-mask and use the appropriate sample sheet to enable demultiplexing.  Please see the CASAVA User Guide for more details on sample sheets and demultiplexing commands.

  • How reproducible is the data?

    We performed experiments to test for reproducible detection of fusion in control samples across multiple replicates and library preps and demonstrated the expected fusions (such as BCR-ABL1 in UHR) are consistently detected as high confidence calls. The exact values of the fusion confidence scores determined by the RNA Fusion Module can very slightly from prep to prep. Therefore, fusions that score just above the default 0.6 score threshold may fall just below in a replicate sample. In this case, it can be helpful to lower fusion score threshold slightly below 0.6.  

  • Can I perform comparisons across large groups of samples for cohort or population analysis?

    No. Comparisons of two or more groups of samples cannot be performed using the application.

  • What is the recommended read length for the libraries?

    The recommended read length is 2 x 75 bp.

  • Will the TruSeq Nano DNA Library Prep Kit work with degraded DNA samples?

    The TruSeq Nano DNA Library Prep Kit requires high quality gDNA as starting material.

  • Is initial cycle indexing possible on the HiSeq?
    No, the system does not support an initial cycle indexing method. To ensure the highest quality data, Illumina recommends and supports a separate indexing read for multiplexed samples.
  • Why does the TruSeq ChIP Sample Prep protocol only select for 250–300 bp fragments after ligation when the starting material is recommended to be fragmented to 200–800 bp in length?

    ChIP DNA samples can be difficult to fragment, and the wider distribution of starting fragment size is chosen to allow for ease of use with a broader range of starting material. Gel-based size selection focuses on a narrow insert size (250–300 bp post-ligation) to allow for increased success with motif finding, which is improved with tighter distributions and smaller fragments. Illumina recommends using smaller starting fragment sizes, if possible, to increase the amount of material in the selected range. Samples with distributions in the 200–800 bp range have been shown to generate successful librarie,s even if the majority of the input material is not in the final size selected.

  • Is the gel size selection step necessary in the TruSeq ChIP Sample Prep protocol?

    Illumina recommends performing the gel size selection step as it is designed to remove unligated adapters, as well as any adapters that might have ligated to one another, and selects a narrow 250–300 bp size-range of DNA fragments for ChIP library construction appropriate for cluster generation.

  • Are there low plexity guidelines?

    Guidelines can be found in the TruSeq Library Prep Pooling Guide.

    This kit was designed for 4-plexing samples. Certain reagents in the kit can run out if running at less than 4-plex.

    When designing low-plexity index pools for single-indexed sequencing, always use at least 2 unique and compatible barcodes for each index sequenced. The following table describes possible pooling strategies for 2–4 samples generated with the adapter index tubes in each set.

    Not all color-balanced pools are listed. Check the color balance using IEM. For more information, see the Illumina Experiment Manager User Guide.

    LT Kit - Any 2-plex, 3-plex, or 4-plex combination of AD002, AD004, AD005, and AD006.

    HT Kit -

    Plexity

    Option

    Indexes

    2

    1

    AD006, AD012

     

    2

    AD005, AD019

    3

    1

    AD002, AD007, AD019

     

    2

    AD005, AD006, AD015

     

    3

    2-plex options with any other index

    4

    1

    AD005, AD004, AD007, AD016

     

    2

    AD002, AD004, AD007, AD016

     

    3

    3-plex options with any other index

  • Are there a minimum number of indices I need to use for TruSeq HT sample prep?
    For dual indexing, at least 2 unique barcodes/indices need to be present for each Index Read. For less than 6 dual-indexed samples, Illumina recommends using single indexing.  Please refer to the pooling guidelines in the TruSeq sample preparation guide for the kit that you are using.
  • When dual-indexing, if I use mismatches=1 for CASAVA, will it take into account 1 mismatch per index or only 1 mismatch total across both indices?

    It will take into account one mismatch per index. The argument in CASAVA is a comma-delimited list of number of mismatches allowed for each read (for example: 1,1). If a single value is provided, all index reads will allow the same number mismatches. the default is 0. The index reads are treated completely separately.

  • What quantification method is recommended before beginning the enrichment procedures?

    Quantify your library using an Advanced Analytical Fragment Analyzer or Agilent Technologies 2100 Bioanalyzer. As an alternative, quantify using PicoGreen.

  • What is the effect of residual contaminating DNA?

    RNA that has DNA contamination results in an underestimation of the amount of RNA used and can impact data quality.

  • Can I import raw intensity files (idats) into the PC Module?

    No, it is not possible to generate a new project in the PC Module directly from idats. The polyploid workflow requires to first generate a genotyping project in the GenomeStudio Genotyping Module from the idats. The genotyping project (.bsc) can then be opened in the PC Module for polyploidy clustering.

  • What applications can I run on the NovaSeq System?

    The NovaSeq Sequencing System is an open platform suitable for a wide range of methods. These methods include, but are not limited to, whole genome, exome, and transcriptome sequencing. Public data sets are available in BaseSpace Sequence Hub.

  • What is ForenSeq DNA Signature Prep used for?

    The ForenSeq DNA Signature Prep Kit is used to determine which STRs and SNP genotypes are present in an individual sample.

  • What consumables and equipment do I need to process ForenSeq DNA Signature libraries in my lab?

    A full list of user supplied items can be found in the Consumables and Equipment section of the ForenSeq DNA Signature Prep Guide or ForenSeq DNA Signature Prep consumables and equipment excerpt. Comparable performance is not guaranteed when using alternate equipment.

  • How many reads are needed per sample for small RNA sequencing?

    This depends on the application. For expression profiling, 1–2M mapped reads is a generally accepted range. For discovery applications, an increase to 10–20M reads may be considered.

     

  • How is it possible to combine multiple samples into one MiSeq FGx sequencing run?

    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.

  • Can I use Nextera DNA or Nextera XT DNA sample prep kits with the Nextera Rapid Capture Enrichment reagents?
    No. The Nextera Rapid Capture Enrichment reagents and protocol have been optimized to only be used together. The kits include all components for both library prep and Rapid Capture.
  • How much space is needed on the output drive?

    Each cycle of a run requires at least 5.4 GB of space. Therefore, a typical 2 x 150 bp mid-output run requires about 1.7 TB of space. For long-term storage, save nonsequencing data to external storage instead of the instrument. Use the Disk Management screen to view available disk space, transfer the run data to external storage, and clear disk space for a new run.

  • What type of library prep is supported on the HiSeq 3000?

    Like the HiSeq 2500, the HiSeq 3000 is designed as an open platform intended to support a broad array of applications. Internally, Illumina has run numerous library prep kits without issue. For sample data demonstrating system performance, see the HiSeq 3000/4000 Sample Datasheets.

    The HiSeq 3000 system is compatible with current paired-end P5 and P7 primers. Single-read and paired-end libraries are compatible with the HiSeq 3000, but must use current adapters. Legacy libraries that are based on the original Illumina single-read adapter do not cluster properly.

  • Which species are compatible?

    The Globin-Zero Gold kits were developed using model organisms. Results can vary depending on the species from which the RNA sample was derived, especially with 5S rRNA removal.

    Visit the RNAMatchMaker tool, www.epibio.com/rnamatchmaker, on the Epicentre website to confirm species compatibly.

  • Can I change the analysis after it has been completed in ForenSeq Universal Analysis Software?

    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.

  • How many lanes are on a NextSeq flow cell?

    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.

  • Can custom primers be used on the NextSeq system?

    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.

  • What is causing my scan on the NextSeq 550 system to fail registration?

    Make sure that the BeadChip is seated flat on the adapter and that the BeadChip is free of dust. Use canned air or other compressed dusting method to clear the debris.

  • What should I do if the copy speed is too slow?

    If you are not copying files to the target server fast enough, consider the following:

    • Illumina recommends a network connection rated at 1 Gigabit or faster. Ask your IT department to upgrade your network.
    • If the target server is nearly full, copying takes much longer. Remove files that are no longer needed from the target server, or copy to a different server.
    • If multiple sequencing instruments copy to the same server, the server drive might not be fast enough to handle the traffic. Consider setting up the instruments to copy to different servers.
    • If multiple sequencing instruments share a connection to the target server, the connection might not be fast enough to handle the traffic. Consider setting up the instruments to use different routes.
  • How much time do typical Rapid runs take?
    Read Length Estimated Rapid Run Time (Hrs)*
    1 x 50 bp no index 9
    1 x 50 bp dual index 11
    2 x 100 bp no index 27
    2 x 100 bp dual index 30
    2 x 150 bp no index 40
    2 x 50 1bp dual index 43
    *Systems with SN < 7000895 will require additional time
  • What factors affect the run-time of the BaseSpace TruSeq Long-Read Assembly and TruSeq Phasing Analysis apps?
    For phasing, the run-time is affected by input short-read volume (GB of short reads). For long-reads assembly, the run-time is affected by GB input, genome complexity, and seeding density.
  • Can I use Zymo beads instead of Sample Purification Beads for the clean up steps?

    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.

  • What is the minimum of amount of samples that I have to prepare using this protocol?

    Both the LT and HT kits contain enough reagents for the indicated number of samples at 4-plex only. You can perform this protocol at different plexities (single-plex, 2-plex) with additional PCR cycles, but some reagents run out when using less than the maximum number of samples.

  • Can I compare data from the TruSeq Stranded mRNA Sample Prep protocol to either the TruSeq RNA Sample Prep or the Directional mRNA-Seq Sample Prep unsupported protocol?

    The data is comparable to TruSeq RNA Sample Prep v2 data. The TruSeq stranded RNA and Directional mRNA-Seq protocols are based on distinct ligation chemistries and the libraries they produce are expected to differ as a result. Direct sample to sample comparison is not recommended for these library types.

  • Can I have both VariantStudio v2.1 and v2.2 installed on the same computer?

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

  • Where do I find the expression data?

    In the RNA-Seq Alignment project, select a sample. In the Important Files for Download section, click the reference gene counts file.

  • What is the recommended cluster density, output, and data quality from HiSeq 1500/2000 systems?considerations with the laser system on the HiSeq or HiScanSQ?
      Rapid (2 Lane) High Output (8 Lane)
    Recommended Cluster Density 750-900 k 750-800 k
    Clusters per lane (PF) 130-150 M 187-210 M
    Per flow cell output (2 x 100 bp) 50-60 Gb 300 Gb
    Per flow cell output (2 x 150 bp) 75-90 Gb No supported
    Bases >Q30 (2 x 50 bp) 85% 85%
    Bases >Q30 (2 x 100 bp) 80% 80%
    Bases >Q30 (2 x 150 bp) 75% Not supported
  • Which version of Illumina Experiment Manager (IEM) is compatible with the NextSeq system?

    A sample sheet is not required for a sequencing run unless you are using the system in standalone mode. Use IEM version 1.8.2, or later, to create a sample sheet.

    If you are connected to BaseSpace, use the BaseSpace Prep tab to record library and indexing information, and to specify other run parameters. When runs are set up on the Prep tab, a sample sheet is not required.

  • What is in the tube labeled "Index adapter" in the TruSeq Targeted RNA Index Kit?
    This tube contains the PCR primers used to amplify the cDNA amplicons. The primer sequences include sequencing primer binding sites and the indices/barcodes.
  • Will the Illumina demonstrated protocols for Direction mRNA-Seq and DSN Normalization work with the TruSeq stranded RNA sample prep kits?

    Illumina has not tested the TruSeq stranded sample prep kits with Illumina demonstrated protocols.

  • Which version of OLB do I need to process my TruSeq data?

    OLB 1.9 or higher must be used for processing control information; earlier versions of OLB will not be able to process controls.

  • What is the minimum number of samples that I can prepare using this protocol?

    There are enough reagent components to prepare 6 batches of 8 samples.

  • Do I need to re-titrate my samples for use with flow cell v3?

    Yes. The new cluster protocol and wider lanes on flow cell v3 make it imperative to re-titrate your samples to optimize cluster densities for higher throughput. qPCR is highly recommended.

  • How is the DNA fragmented?
    Nebulization is a very reproducible process that produces random fragmentation. Alternative fragmentation methods may be more appropriate depending on sample availability. Other available technologies include sonication, ultrasound, and hydroshearing.
  • What workflow do I use in the Illumina Experiment Manager for creating a sample sheet for my Nextera Rapid Capture Enrichment project?
    Use the Enrichment workflow to create a sample sheet formatted for analysis of Nextera Rapid Capture Enrichment data. For more information, see the IEM Nextera or TruSight Quick Reference Card for instructions on creating your sample sheet.
  • Does paired-end resynthesis happen before the Index 2 Read for dual-indexing?

    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).

  • Do I need to prime SBS reagents in HiSeq v4 run mode?

    Yes. Priming SBS reagents is required. The control software prompts for this step, which takes about 15 minutes.

  • When do I use a PhiX spike-in for sequencing?

    For samples with an unbalanced base composition, Illumina recommends using a high-concentration spike-in of PhiX control lane to create a more diverse set of of clusters. Unbalanced samples contain genomes with AT or GC content of less than 40% or greater than 60%. PhiX Control v3, Catalog # FC-110-3001, is a balanced and diverse library with a mean insert size of approximately 375 bp, corresponding to 500 bp library size if visualized on a Bioanalyzer. For more information, see Using a PhiX Control for HiSeq Sequencing Runs.

  • What sequencing redundancy is recommended?
    This depends on the size of the organism you are trying to resequence. For whole-genome resequencing, a 25-fold over-sampling should be adequate. For targeted resequencing involving mixes of many PCR products, 75-fold over-sampling will correct for the inability to mix the PCR products at a 1:1 ratio. Illumina sample prep shows no systematic bias. In sequencing the X chromosome, we achieved 16-fold average cover­age with all sequenceable bases covered at least twice.
  • How does the Isaac human WGS workflow compare to BWA / GATK in terms of sensitivity and specificity?

    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. 

     

    Conflicts

    Conflict Rate

    Sensitivity

    Isaac

    6318

    0.139%

    94.5%

    BWA+GATK

    5315

    0.126%

    95.8%

     

     

     

    Total number of SNP conflicts, SNP conflict rate, and sensitivity (% of non N reference sites called) of Isaac and BWA+GATK

  • What is the difference between the TruSeq Synthetic Long-Read DNA library prep long-read and phasing workflows?

    For the Phasing workflow—

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

    For the Long-Read workflow—

    • 3 fg library is required per well during the Long Range PCR procedures.
    • 21 and 26 PCR cycles are run during the Long Range PCR procedures.
    • BaseSpace TruSeq Long-Read Assembly is used for analysis.
  • Can I use the TruSeq HT adapter plate to generate just a few samples at a time?

    The adapter plate is single-use per well and Illumina recommends that it not undergo more than four freeze/thaw cycles. For this reason, Illumina strongly recommends processing at least 24 samples at a time with the TruSeq HT 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.

  • Does Illumina recommend using the TruSeq Stranded Total RNA Sample Prep Kit with Ribo-Zero Globin on any sample other than blood?
    The Ribo-Zero Globin kit has been specifically tailored for blood samples from human, mouse, or rat. In addition to removing cytoplasmic and mitochondrial rRNA, it will also deplete globin mRNA. Illumina recommends using the Ribo-Zero Gold kit for samples other than blood.
  • Does Illumina recommend checking final NeoPrep libraries on a Bioanalyzer ?

    A library quality check on a Bioanalyzer is optional. See the appropriate library prep reference guide for library trace examples.  

  • Is it recommended to normalize mRNA and miRNA together in the same project?
    Illumina recommends normalizing mRNA data separately from miRNA data.
  • How does this protocol handle ribosomal RNA contamination?

    Because it enriches the coding transcriptome, the ribosomal RNA is washed away during the wash procedures.

  • Can I run both single read and paired-end recipes with TruSeq HT dual-indexed libraries?

    Yes. However, for optimal coverage, Illumina recommends sequencing stranded RNA libraries with paired-end chemistry.

  • Can I run both single read and paired-end recipes with TruSeq HT dual-indexed libraries?

    Yes. However, for optimal coverage, Illumina recommends sequencing stranded RNA libraries with paired-end chemistry.

  • Is there a gel-free TruSeq Sample Prep protocol available?
    • TruSeq DNA has a gel-free protocol option (targeted resequencing only).
    • The TruSeq RNA protocols have a gel-free workflow.
  • Is HCS v2.2 available for non-upgradeable systems?

    Yes. HCS 2.2 is available for all customers.

  • How do Nextera DNA Sample Preparation Kits work?

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

  • Will I need to re-validate HCS 1.5/RTA 1.13 if upgrading from HCS 1.4/RTA 1.12?
    Validation of HCS 1.5/RTA 1.13 should not be required if you are upgrading from HCS 1.4/RTA 1.12. Changes in RTA do not affect data quality, and changes in HCS updated the user interface to enable dual indexing. Please refer to the HCS 1.5 Release Notes for additional information about new features in this software package.
  • Can I perform sample to sample comparisons?

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

  • How do you ensure that different adapters are ligated to each end of a DNA fragment using the TruSeq DNA PCR-Free protocol?
    Illumina's proprietary method ensures ligation of two different adapters in the required orientation to opposing ends of a DNA fragment. Fragments without the proper adapters ligated at both ends may remain due to the absence of PCR enrichment that selects for completed products. Ligation is ensured by performing a qPCR step at the end of the protocol. qPCR will only quantify libraries that have complete adapter ligations and will cluster and sequence. Other methods will quantify fragments that may not have adapters at both ends and will thus not generate clusters or sequence. Quantification by methods other than qPCR will be inaccurate.
  • What molecules are removed with the TruSeq Stranded Total RNA Sample Prep Kit with Ribo-Zero Plant?
    The Ribo-Zero removal mix targets cytoplasmic (25S, 18S, 5.8S, and 5S rRNA), Chloroplast (23 S, 16S, 5S, and 4.5S), and mitochondrial rRNA (18S and 5S).
  • How many cycles should be used during the Index Read for single-indexed libraries?

    Index reads for single-read libraries use 7 cycle reads. Illumina does not support 6 cycle index reads for single-indexed libraries.

  • What kits are available and how many enrichment reactions can be processed with each kit?

    This kit is available in a Set A and a Set B. Each kit contains enough reagents to prepare up to 48 samples with 12 unique indexes. When used together, sets A and B allow for pooling up to 24 samples using the 12 different indexes in each kit.

  • Which version of Off-Line Basecaller (OLB) should I use to reprocess dual-indexed data from cif files?

    Use OLB 1.9.3 or later to process data generated on HiSeq 2000, HiSeq 1000, or HiScanSQ. Use OLB 1.9.4 to process data generated on the Genome Analyzer.

  • Can I use only 1 of the indexes of a dual-indexed library?

    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.

  • What software can be used to analyze the libraries?

    The BaseSpace RNA-Seq Alignment App performs alignment and analyzes for gene fusions with STAR or TopHat. STAR is recommended for optimal fusion calling.

    The BaseSpace TopHat Alignment App can also be used for alignment and fusion calling. The BaseSpace Cufflinks Assembly & DE App can be used to run differential expression analysis.

    Third party analysis tools are also available.

  • Does BaseSpace Variant Interpreter automatically assign variant classification?

    No. For germline variants, the software uses a simple rule set to predict the classification:

    • Automatically ranks and prioritized variants based on their annotations, predicted consequence (loss of function, missense, or noncoding) and population allele frequency (using the highest frequency from ExAC, 1000 Genomes, and EVS).
    • Variants are typically assigned the ClinVar pathogenicity unless the variant is greater than 5% population frequency, in which case they are assigned Benign per ACMG guidelines.
    • Variants without ClinVar entries that are common (over 1% population frequency) are assigned Likely Benign.
    • Variants without ClinVar entries that are uncommon (less than 1% population allele frequency) are assigned Likely Pathogenic if loss-of-function (frameshift, stop-gained or essential splice), VUS if a missense or near-splice, and Likely Benign if noncoding.

    This pathogenicity autoscoring is only a suggestion. Review these predictions, the provided annotations, and all evidence for the variant to assign your final interpretation.

  • How often do I need to replace a wash flow cell?

    Replace a wash flow cell when it becomes visibly worn. Wash flow cells do not contain any chemistry, so consistent replacement is not necessary.

  • After what cycle will TruSeq control information be available?
    • Control information for a read is processed after alignment (cycle 25) but is not reported until cycle 52. For an indexed read, control information is reported after cycle 52 of R2.
    • If performing a run shorter than 52 cycles, you should see control reporting that is accurate for all controls except CTE, which will report counts in all band sizes.
  • What data can I expect from the ChIP-Seq workflow on the MiSeq?

    The ChIP-seq workflow generates demultiplexed FASTQ files.

  • How long does the TruSight DNA Amplicon assay take?
    The assay takes less than eight hours total, with 2–3 hours of hands on time. TruSight DNA Amplicon libraries are ready to run on an Illumina sequencer without any further manipulation.
  • How do I enable positive sample tracking?

    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).

  • What do the status bar colors indicate on the front of the MiSeq?
    Green indicates the instrument is ready to run, blue indicates the instrument is running, and orange indicates the instrument needs attention.
  • What is the typical library size distribution?

    The size of the final product is approximately 250-300 bp. A larger fragment size is expected for good FFPE RNA (> 350 nt), while a smaller fragment size is expected for poor FFPE RNA.

  • Can I customize a HiSeq recipe?
    With HCS v1.3 and later, you can customize a recipe to contain any number of reads. Reads can be indexed or non-indexed. However, Illumina does not guarantee the performance of custom recipes. Contact your Illumina Technical Support if you need assistance creating custom recipes.
  • Are original cBot manifolds compatible with cBot 2?

    No, original manifolds do not align correctly on the new instrument.

  • What is included in this kit?

    This kit include all the reagents needed for generating complete tagmented and enriched libraries ready for sequencing. The kits also include magnetic SPRI beads for sample clean-up steps. For more information, see the Kit Contents section of the library prep reference guide.

  • Where is barcode information stored for the sample tracking workflow?

    Barcode information is recorded and stored in the run data file (*.xml). This file is written to a preferred network location, which you can configure from the LIMS tab.

  • What instruments support the TruSeq Amplicon - Cancer Panel 2.0?

    Illumina recommends running TruSeq Amplicon - Cancer panel 2.0 on the MiSeq. However, this assay is also compatible on the NextSeq, and HiSeq. Perform analysis using the TruSeq Amplicon BaseSpace App or MiSeq Reporter TruSeq Amplicon Workflow.

  • Which regions are targeted with the oligos in this kit?

    The oligos target all exons of every gene.  Download the Target Regions file for more information.

  • How many libraries can I prepare per NeoPrep run?

    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.

  • Can I perform family-based genetic disease analysis in VariantStudio?

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

  • Are there any performance differences (in terms of sequencing coverage) for GC rich amplicons?
    Although most amplicons of interest will not likely be high GC-content, please note that coverage of high GC-content amplicons may be more variable as compared to other amplicons.
  • How long does it take to perform cluster generation and primer hybridization on the MiSeq?
    This step takes approximately 70 minutes.
  • Will this assay detect chromosomal translocations that do not generate a fusion gene?

    Chromosomal translocations resulting in overexpression or deletion of a transcript can be reflected in gene expression levels but would not create a fusion gene.  The Local Run Manager RNA Fusion module is not designed for detection of gene expression changes. To detect these changes, the RNA-Seq Alignment App in BaseSpace Sequence Hub is recommended. In addition, it is recommended to confirm these findings in DNA.

  • Can I import custom 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.

  • I get the following error message, what should I do? ERROR: previous run [13_01_39_04_09_08] didn't finish
    One option is to clean all your data and run your command CMD again ˜/ CMD --target=allClean ˜/ CMD
  • Is a reagent blank included in the ForenSeq DNA Signature Prep Kit?

    No, the reagent blank is made by the kit user.

  • What flow cell should be used with TruSeq ChIP libraries?

    TruSeq ChIP libraries can be sequenced using either Single Read or Paired End flow cells.

  • What Illumina sequencing platforms are compatible with Nextera?
    Nextera is compatible with all Illumina sequencing platforms: MiSeq , HiSeq 2000, HiSeq 1000, HiScanSQ , and GAIIx.
  • Can a USB device be plugged into the MiSeq during the run?
    Illumina recommends that you wait until the completion of a run before inserting a USB device into the MiSeq.
  • I see lower coverage of GC regions than I was expecting with the TruSeq Nano DNA Library Prep protocol. What could be a factor?

    Coverage of GC regions can be impacted by the model, settings, and performance of the thermal cycler used. Illumina has validated the Bio-Rad DNA Engine Tetrad 2, the Bio-Rad S1000, and the MJ Research PTC-225 DNA Engine Tetrad. Other thermal cyclers may differ in their performance across the genome.

  • Are there safe stopping points in the sample prep process?
    Yes. You can safely stop this protocol after the ethanol precipitation or any Qiagen purification step. Store the samples at -15° to -20°C.
  • How many genes does the TruSeq Amplicon - Cancer Panel target?

    Mutational hotspots from over 48 genes associated with cancer are targeted with 212 amplicons.

  • What type of library prep methods are supported on the HiSeq X system?

    Use either the TruSeq Nano DNA (HT or LT) or the TruSeq DNA PCR-Free (HT or LT) library prep kits. An insert size of 350 bp or 450 bp is supported.

    For more information, see the HiSeq X Ten system kit page.

    Illumina does not provide support for any other library prep methods or alterations to Illumina protocols, or guarantee system performance with other than the supported library prep methods on the HiSeq X.

  • How much input genomic DNA is required for the TruSight One workflow?
    50 ng of input genomic DNA is required. It is important to quantify the input genomic DNA to generate a high-quality library of the correct size.  Illumina highly recommends using a fluorometric-based quantification method for the input genomic DNA. For more information, see the DNA Input Recommendations section of the TruSight One Library Prep Guide.
  • Is there a lower limit of the number of amplicons to use in my pool?
    To obtain higher quality sequencing data, Illumina recommends sequencing samples with high diversity and avoiding monotemplate stretches during sequencing. Low diversity can occur with pools of one or a few amplicons. Additionally, it is important to maintain color balance for each base of the read or index read being sequenced, otherwise sequencing could fail due to registration failure. Please refer to the MiSeq System User Guide (part # 15027617) for recommendations on low diversity libraries or the Nextera XT User Guide (part # 15031942) for information on low plexity pooling guidelines.
  • What BeadChips are supported on the NextSeq 550 system?

    Supported BeadChips include the CytoSNP-850K, HumanCytoSNP-12, and HumanKaryomap-12.

  • Is there a limit to the amount of contiguous sequence that can be designed against in DesignStudio?

    There is a 24 kb limit of contiguous sequence that can be entered for a given target region so as not to overwhelm the design server memory resources. For designs spanning large stretches of DNA, Illumina recommends Nextera Rapid Capture Custom Enrichment, a hybridization-based enrichment for targeted resequencing.

  • Are TruSeq Custom Amplicon, TruSeq Amplicon Cancer Panel, and Nextera XT applications supported to run on the HiSeq?

    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.

  • What is the recommended loading concentration for sequencing NeoPrep libraries?

    NeoPrep libraries are ready for cluster generation. If you have performed quantification as part of the NeoPrep run, see the suggested clustering recommendations on the Requirements & Compatibility support page for the appropriate library prep kit.

  • How many fusion-supporting reads are required to call a fusion?

    The BaseSpace RNA-Alignment App with the STAR aligner may call a fusion if there are at least 3 unique reads that meet all the quality metrics, including the following threshold filters.

    - Split Reads + Paired Reads ≥ 3

    - Alt/Ref Reads ≥ 0.01

    - Fusion Contig Align Length (bp) > 16

    - Break-end Homology (bp) ≤ 10

    - Alternative Local Contig Align Fraction < 0.8

    - Coverage after fusion (bp) ≥ 100

    However, a high number of nonfusion supporting reads in that region would be expected to cause ‘noise’ that can affect fusion calling.

  • Can VariantStudio be used in a clinical lab, such as a CLIA lab?
    Yes, but only after validating the software according to Institution, Local, State, and Federal guidelines before using the Illumina VariantStudio software.
  • If a network outage to the central file server storing our runs occurs, can the HiSeq 4000 cache an entire 2 x 150 run?

    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.

  • What is the success rate for TruSeq Targeted RNA Expression designs?
    Illumina internal testing finds that > 90% of non-validated assays correlate with RNA-Seq data. Assays marked validated have been shown to give results consistent with fold changes observed by RNA-Seq in testing across human tissues.
  • What are my options for analyzing NovaSeq sequencing data?

    BaseSpace Sequence Hub is the preferred analysis solution for the NovaSeq Sequencing System. On-instrument analysis is not available. For third-party and user-developed analysis packages, you can use the bcl2fastq2 converter.

  • Why are the sequencing intensity profiles so wobbly in the first ~15 cycles of my run?

    This is a normal characteristic of the tagmented libraries and relates to the sequence context at the tagmentation site.

  • Which reference genome was used to generate probes?

    UMD 3.1 of Bos Taurus 

  • How do I obtain the cluster and manifest files required to scan a BeadChip on the NextSeq 550 system?

    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.

  • What is the recommended minimum number of reads per sample and sequencing format for ChIP-Seq?
    For studies targeting transcription factors, we recommend between 5 and 15M 1×35–1×50 reads per sample. For studies targeting histone modifications, we would recommend between 50–90M 1×35–1×50 reads. 
  • What is the difference between TruSeq Methyl Capture EPIC and TruSeq DNA Methylation?

    TruSeq DNA Methylation is used for whole genome bisulfite sequencing and uses an entirely different chemistry than TruSeq Methyl Capture EPIC.

  • Can I adjust the hybridization times?

    No, there is no range of hybridization times. The protocol has been optimized for 30 minutes for each hybridization

  • Are there any safety considerations with the laser system on the HiSeq or HiScanSQ?
    The HiSeq and HiScanSQ are Class 1 laser instruments as evaluated by IEC 60825-1 Edition 1.2. Under normal operation, the operator is not exposed to laser light.
  • Can I process a low-diversity sample on a HiSeq 3000?

    True low diversity samples, such as single amplicon and 16S samples, are not expected to produce quality results. However, spiking in a well-balanced genome such as PhiX improves performance.

  • What applications can I run on a MiSeq?

    The MiSeq system is ideal for amplicon sequencing, targeted resequencing, small genome sequencing, and clone checking. It is capable of performing 16S ribosomal RNA gene sequencing, ChIP-Seq (TF Binding), and small RNA sequencing.

  • Will TruSeq v3 reagents continue to be sold?
    Yes. There are currently no plans to discontinue the sale of TruSeq v3 kits.
  • Can I start with mRNA for TruSeq Targeted RNA Expression?
    Use 0.5–2.5 ng of optimized mRNA input. However, starting with mRNA input has not been tested and is not currently supported.
  • What operating system runs on the NextSeq computer?

    The operating system on the NextSeq computer is Windows 7.

  • What is an amplicon and what is the size of an amplicon from TruSight DNA Amplicon libraries?
    An amplicon is a fragment of DNA that is produced from an amplification event such as PCR. For TruSight Myeloid libraries, amplicon sizes are fixed at a length of 225–275 bp.
  • How do I prevent the magnetic beads from freezing in storage?

    If frozen, the magnetic beads do not work. Confirm that the beads were not frozen when they arrived in the lab, and that they were properly stored at 2°C to 8°C.

  • What methods of shearing are supported by TruSeq Nano DNA Library Prep?

    The protocol is optimized for shearing using Covaris fragmentation. Other methods are not supported or tested by Illumina and may result in low yield, unexpected size distributions, or sample failure.

  • What are the supported insert sizes on the NextSeq system?

    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.

  • Does the minimum breakpoint distance start from the end of the 1st gene to the start of the 2nd gene?

    Yes. The shortest possible distance between the two genes, either the start or end coordinate, is used to determine the distance between the genes. It is not determined by the breakpoint position.

  • Can the ChIP-Seq protocol be used to make libraries from low starting amounts of non-ChIP DNA?
    Yes, although this requires some adaptation of the protocol. The ChIP-Seq protocol assumes that the input DNA is already fragmented. If the intended starting material is genomic DNA, it will need to be fragmented before use with this protocol. Fragmentation methods such as Covaris offer higher recovery and a tighter size distribution than nebulization, and are preferred for this sort of application. At least 10 ng fragmented DNA should be used.
  • What level of indexing is supported for TruSeq DNA and RNA Sample Prep v1 and v2/LT?
    • TruSeq DNA and RNA Sample Prep v1 kits support 12-plex per lane and 96-plex per flow cell.
    • TruSeq DNA v2/LT and RNA Sample Prep v2 kits support 24-plex per lane and 192-plex per flow cell.
  • What is the estimated genomic pull down region from a probe designed in DesignStudio for Nextera Rapid Capture Custom Enrichment?
    The estimated region is 280 bp total with the probe at the center.
  • Does the MiSeq reagent cartridge contain sequencing primers for all applications?
    Yes. The MiSeq reagent cartridge contains sequencing primers for TruSeq DNA and RNA samples, TruSeq Small RNA samples, TruSeq Custom Amplicon libraries, and Nextera DNA samples.
  • How many empty ports are available on the reagent cartridge?
    Three reservoirs on the MiSeq reagent cartridge are available for user-supplied custom primers. For more information, see Using Custom Primers on the MiSeq (part # 15041638).
  • What tools are available for data analysis?

    Use Sequence Genotyper to convert variant call files (VCF) to a genotype call file.

  • Are TruSeq Cluster Kits v3 and SBS Kits v3 available for use on the Genome Analyzer?

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

  • Do I need kits, in addition to TruSeq HT sample prep, in order to sequence TruSeq HT dual-indexed libraries?

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

  • What is deconvolution?
    It is the ability to distinguish between two or more clusters that are in close proximity to each other.
  • What is the typical library size distribution for my final libraries?

    The expected size range for final libraries should be from ~250 bp to ~1 kb and the expected median insert size is 180-200 bp.  See the reference guide for an example of a Bioanalyzer trace.

  • What insert size should I use?
    Between 150 bp and 200 bp is the ideal size range for single-read libraries. The final product of the sample prep process will be about 100 bp longer than the size of the cDNA insert due to the additional length of the Illumina linker and primer regions.
  • What are the safe stopping points in the protocol?

    There are 3 safe stopping points in the protocol. The safe stopping points are after the following steps: Fragment DNA, Amplify Enriched Library, and Clean Up Amplified Enriched Library.  For storage details, see the reference guide.

  • If I am running a software version older than HCS 1.4.8, do I need to serially upgrade to 1.4.8 prior to installing HCS 1.5?

    You can upgrade directly to HCS1.5/RTA1.13 from HCS1.4/RTA1.12. If you are running an older version of HCS, please contact Illumina Technical Support for assistance in upgrading.

  • Does this kit use dual index adapters and how long are the indexes?

    Yes, this kit contains dual index adapters - 24 i7 indices and 2 i5 indices.

    The i7 index is 6 bp and i5 index is 8 bp. Although numbering of indexes seems similar to other kits, the index sequence is different and can be found in the reference guide.

  • Can I run TruSight DNA Amplicon libraries on instruments other than the MiSeq?
    Yes. TruSight DNA Amplicon libraries can be run on any Illumina sequencing platform but only the MiSeq provides a built-in analysis solution—MiSeq Reporter. NextSeq and HiSeq users can perform analysis using BaseSpace or BaseSpace Onsite, their own pipelines, or third-party tools. Amplicon workflows are expected mid-2014.
  • How does the Ribo-Zero portion of the TruSeq Stranded Total RNA Sample Prep protocol work?

    TruSeq Stranded Total RNA Sample Prep Ribo-Zero kits remove ribosomal RNA (rRNA) using a hybridization/bead capture procedure that selectively binds rRNA species using biotinylated capture probes. The probe:rRNA hybrid is then captured by magnetic beads and removed using a magnet, leaving the desired rRNA-depleted RNA in solution.

  • Can I compare data between the TruSeq small RNA protocol and earlier Illumina small RNA protocols?

    As with any molecular biology techniques, a change in protocol will give a change in absolute results. We do not recommend comparing absolute counts (or counts normalized to total counts) between different preparation protocols. However, comparison of fold change between the two protocols should yield good correlation.

  • Can I combine HiSeq v4 SBS kits to achieve read lengths longer than what is provided in a kit?
    Yes. See “Preparing SBS Reagents” in the HiSeq 2500 and HiSeq 1500 user guides for a list of how many SBS kits can be combined for supported run lengths.
  • Can I use cBot cluster kits on the cBot 2?

    Yes. All cluster kits intended for use on the cBot are also compatible with cBot 2.

  • What is the recommended amount of RNA library for enrichment?

    Use 200ng of each RNA library.

  • What is the error rate on MiSeq?

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

  • Are FFPE samples supported for TruSeq Amplicon - Cancer Panel?

    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).

  • Does this assay use the same PCR/sequencing primers as Nextera?

    No. The assays use different oligos.

  • What is the difference between TruSeq DNA and RNA Sample Prep v1 and v2/LT Kits?
    • No changes to workflow, increase in index capability
    • Fill volumes and new consumables to support automation
    • Each kit contains 12 of 24 unique indexes and each index reaction sufficient for eight individual samples
  • Does Illumina VariantStudio support TruSight Tumor data?

    Yes. The Amplicon-DS workflow in the MiSeq Reporter software is used for the alignment and variant calling of TruSight Tumor data. The resulting VCF files can be imported into VariantStudio. Illumina recommends that you use the merged VCF file from the Amplicon-DS workflow. In these files, the variant call information from both the forward pool and reverse pool has been merged.

  • What sample types has this assay been tested with?

    This assay has been tested with RNA from blood, bone marrow, and FFPE tissues.

  • Can I use Sequencing Analysis Viewer to view data from a NovaSeq run?

    Yes. Sequencing Analysis Viewer (SAV) v1.11 or later is required to view data from a NovaSeq System run. You can download SAV for use on a computer or use BaseSpace Sequence Hub.

  • What do I need to run MiSeq v2 chemistry?
    Using reagents provided in the MiSeq Reagent Kit v2 requires the recipes and RFID recognition settings in MiSeq Control Software (MCS) v2.0. To download a copy of MCS v2.0, see the MiSeq System downloads page.
  • Can I perform a primer rehyb on the MiSeq?
    No, a primer rehyb is not possible on the MiSeq.
  • What quality control method is recommended for the final libraries?

    Assess the final library quality with either an Advanced Analytical Technologies Fragment Analyzer using a NGS Fragment Analysis Kit or Agilent Technologies 2100 Bioanalyzer using a DNA 1000 chip.

  • How many swaths and tiles are on a 2-lane rapid run flow cell?

    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.

  • Can I reorder the same TruSeq Targeted RNA Expression oligo pool multiple times?
    Yes. Your orders are saved in DesignStudio and can be reordered or used for an add-on project. If you need a large number of samples (> 10,000), contact Inside Sales.
  • Can I do paired-end sequencing with mRNA-Seq libraries?
    mRNA-Seq libraries are built with paired-end adapters and amplification primers. If you run them on paired-end flow cells, two paired runs of sequence data will be generated. However, these paired sequences can span unknown splicing junctions, which complicates alignment to a reference transcriptome. For this reason, Illumina does not support this application. However, users with bioinformatics experience can create their own alignment methods to handle these paired reads.
  • How was the content for this panel chosen?

    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).

  • What if I want to do an experiment that does not fit into one of the MiSeq Reporter analysis workflows?
    You can set up your sample sheet to use the GenerateFASTQ workflow, which generates FASTQ files and does not proceed to further analysis. FASTQ files can be exported for analysis by a third-party software.
  • Do I need a manifest to sequence on the MiSeq system?

    A manifest is required to sequence on the MiSeq. The manifest file is available on the Downloads page of the support pages.

  • Are FFPE samples supported for TruSight DNA Amplicon libraries?
    No. FFPE DNA is not supported.
  • What is the library loading volume and concentration required for sequencing on the NextSeq system?

    Using NextSeq Control Software v1.3, or later, the loading volume is 1.3 ml and the loading concentration is 1.8 pM.

    For more information, see NextSeq System Denature and Dilute Libraries Guide (15048776).

  • Why do my designed amplicons include SNPs in the probe regions when Avoid SNPs is turned on?

    When Avoid SNPs is turned on, DesignStudio considers the location of polymorphisms and avoids them when possible. If this is not possible, it will place the probes in regions that do not interfere with their binding.

  • Are TruSeq HT dual-indexed libraries single read or paired-end?
    All TruSeq libraries are paired-end libraries.
  • What is the purpose of the double-sided SPRI after tagmentation and before PCR?

    This size selection ensures that only fragments of the desired size go into PCR, optimizing the enrichment for the best results.

  • Can the TruSeq RNA libraries be used with the DSN Normalization protocol?

    The DSN Normalization protocol was optimized for pre-TruSeq libraries. The TruSeq RNA libraries are not readily amenable to the DSN treatment. There are two primary problems:

    • The Elute-Prime-Fragment mixture is not optimized for non-poly(A) RNA.
    • The PCR primers in TruSeq RNA were optimized for a high yield output. The DSN provides much lower yield due to the duplex-specific nuclease activity, so the PCR primers are not efficient to amplify the DSN-treated TruSeq RNA libraries.
  • Can the TruSeq RNA libraries be used with the DSN Normalization protocol?

    The DSN Normalization protocol was optimized for pre-TruSeq libraries. The TruSeq RNA libraries are not readily amenable to the DSN treatment. There are two primary problems:

    • The Elute-Prime-Fragment mixture is not optimized for non-poly(A) RNA.
    • The PCR primers in TruSeq RNA were optimized for a high yield output. The DSN provides much lower yield due to the duplex-specific nuclease activity, so the PCR primers are not efficient to amplify the DSN-treated TruSeq RNA libraries.
  • Can I run TruSeq HT libraries with Nextera libraries on the same lane or same flow cell? Are the indices the same or different?

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

  • Which reference genome version was used to design this kit?

    Human UCSC version hg19, which is the same as Genome Reference Consortium build 37 (GRCh37).

  • What method should I use to quantify my total RNA input?

    Use a fluorometric assay (ie Ribogreen or Qubit) to accurately quantify the total RNA input. For more information, see the RNA Input Recommendations section in the reference guide.

  • How many TruSeq Amplicon Cancer Panel samples do you recommend that I pool together to sequence on the MiSeq?
    For 300 cycle MiSeq v2 kits, we recommend that up to 60 samples per MiSeq run are pooled. When this guidance is followed, >1000x mean coverage per amplicon per sample should be achieved in runs with 15M clusters passing filter. The new MiSeq v3 kits can deliver >25M clusters passing filter and can accommodate 96 cancer panel samples per MiSeq run to achieve the same >1000x mean depth of coverage.
  • How does dual indexing work?
    For paired-end flow cells, dual indexing now requires 23 additional cycles of sequencing - eight cycles for the Index 1 (i7) Read, eight cycles for the Index 2 (i5) Read, plus seven non-imaging, chemistry-only cycles at the beginning of the Index 2 (i5) Read. For single-read flow cells, dual indexing only requires 16 additional cycles of sequencing - eight cycles for the Index 1 (i7) Read and eight cycles for the Index 2 (i5) Read.
  • How does dual indexing work?
    For paired-end flow cells, dual indexing now requires 23 additional cycles of sequencing - eight cycles for the Index 1 (i7) Read, eight cycles for the Index 2 (i5) Read, plus seven non-imaging, chemistry-only cycles at the beginning of the Index 2 (i5) Read. For single-read flow cells, dual indexing only requires 16 additional cycles of sequencing - eight cycles for the Index 1 (i7) Read and eight cycles for the Index 2 (i5) Read.
  • What are the recommended loading concentrations, number of libraries per flow cell, and reads per sample?

    Sequencing Instrument

    Loading Concentration (pM)

    Number of Libraries Per Flow Cell

    Reads Per Sample (M)

    MiniSeq

    2-2.6

    8

    9

    MiSeq (v3 reagents)

    20-30

    8

    3

    NextSeq

    2-3 24
    18
  • How can I compensate for samples that have variable concentrations, quality, or integrity?

    This situation is a challenge for any system trying to quantify gene expression levels. Variable quality is more challenging than variable compensations for the mRNA-Seq Assay. As described in the question about RNA degradation, we recommend against comparing results across samples that have a large range of quality and integrity, because there will be a large effect on the read coverage across the full length of the transcripts.

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

  • What sequencing primers do I need to sequence my TruSeq Synthetic Long-Read DNA libraries?
    Use the TruSeq Dual Index Sequencing Primer Box, Paired-End (Catalog # PE- 121-1003) when performing cluster generation with the TruSeq PE Cluster Kit v3. If you are using TruSeq PE Rapid Cluster kit or HiSeq PE Cluster Kit v4, no additional primers are required.
  • Is TruSeq Targeted RNA Expression oligo design biased to the 3' end of mRNA?
    No, DesignStudio assays target splice junction sites across the entire transcript. Additional designs are available for cSNPs or gene fusions. The following databases were used for assay designs:

    Species

    Database

    Human junction

    hg19 + refseqs transcript model

    Human cSNP

    dbSNP build 137, common SNPs ≥ 1% MAF

    Mouse junction

    mm9, mm10 + refseq transcript model

    Mouse cSNP

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

    Rat junction

    rn5,rn4

    Rat cSNP

    dbSNP build 125 for rn4

  • Where can I find technical information describing HiSeq v4 data compression options?
    See the Illumina whitepaper, Reducing Whole-Genome Data Storage Footprint, which is available on the Illumina website.
  • What quantification method is recommended for the final libraries?

    Quantify your libraries using qPCR according to the Illumina Sequencing Library qPCR Quantification Guide.

  • Can other thermal cyclers be used with this protocol?

    Coverage of GC regions can be impacted by the model, settings, and performance of the thermal cycler used. Illumina has validated the Bio-Rad DNA Engine Tetrad 2, the Bio-Rad S1000, and the MJ Research PTC-225 DNA Engine Tetrad thermal cyclers. Other thermal cyclers can differ in their performance across the genome.

  • What are the insert sizes for TruSeq DNA PCR-Free libraries?

    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.

  • What is the recommended input amount for TruSeq ChIP sample prep?

    Illumina recommends 5–10 ng of ChIP-enriched, fragmented DNA.

  • What sequencing length is recommended?

    The insert fragments will have a median size of around 150 bp. Therefore, 2 x 76 cycle sequencing is recommended. Longer sequencing lengths show diminishing returns in terms of coverage.

  • Can Covaris settings be altered to produce fragments of different lengths?

    The protocol has been optimized for fragments of 190 bp insert size. Altering this length could cause sample loss during bead size selection steps.

  • What is the typical quantity of the final libraries?

    The expected quantity is 3-12 ng/µL resulting in 10-40 nM. 

  • What are the advantages of the TruSeq Nano DNA Library Prep Kit?

    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.

  • Can I import data from non-Illumina platforms into GenomeStudio software?
    No. GenomeStudio software is intended for use only with Illumina data.
  • I am currently finishing a project using MiSeq v2 reagents. Can I upgrade my software to MCS v2.3 and still finish my project with v2 reagents?

    Yes. The new MiSeq software package is backward compatible with v2 reagents. Using the RFID feature, the MiSeq automatically recognizes which kit version is loaded for the run and chooses the appropriate Q-table. There are no changes to v2 workflows.

  • Are the controls the same in the TruSeq DNA and RNA sample prep kits?

    The same controls are used, but at different concentrations.

  • What kind of quality scoring method does Illumina use?

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

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

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

  • What is the minimum data required and maximum sequencing data used for TruSeq Synthetic Long-Read DNA phasing analysis and long-read assembly?
    A minimum of 30 GB of short-read data is recommended. A warning is given for data sets < 30 GB. Analysis of < 30 GB data may result in poor phasing or assembly results. The maximum input is 115 GB of short read data. A warning is issued if this limit is exceeded.
  • What is the maximum amount of input RNA that I can use for TruSeq Targeted RNA Expression?
    Illumina has tested up to 500 ng of high quality RNA in starting material. However, no improvement in yield is seen in the final amount of RNA generated beyond 200 ng of starting material for high quality RNA.
  • Are clean‐ups/size selection steps necessary?

    The protocol includes one Zymo cleanup step (following "tagmentation") and one Ampure XP size selection step (following limited cycle PCR). The final library should have a median insert size of ~250-300 to support long paired end 2x150 read lengths on the MiSeq system.

  • Can I use a thermal cycler in place of a microheating system for tagmentation in the Nextera Rapid Capture Enrichment protocol?
    Incubation temperature and times have been established based on using the required MIDI plate and TruTemp microheating system. However, efficient tagmentation can take place using a thermal cycler and compatible 96-well plate. However, because of the additional sample transfers and requirement for manual pipette mixing, some degree of sample loss can incur which can negatively impact library diversity.
  • Can I remove targets from Illumina predesigned Nextera Rapid Capture Enrichment panels?
    The add-on workflow does not allow removal of targets from fixed content panels or previously ordered custom designed panels.
  • I'm getting error messages running CASAVA. What do they mean?
    Please send an email to Illumina Technical Support (techsupport@illumina.com) that includes the nohup output file and the command line you used. One of our bioinformatics specialists will respond.
  • What is the difference between a lane and a channel?
    The terms lane and channel are sometimes used synonymously in regards to the eight lanes of a flow cell. However, the term channel may also refer to a color channel on the Genome Analyzer (four colors corresponding to the four bases A, C, G, or T).
  • Which gene fusions for TruSeq Targeted RNA Expression are included in DesignStudio?

    Click here for a list of the gene fusions that are included in TruSeq Targeted RNA Expression.

  • In VariantStudio v2.2, can I choose to continue to use the previous annotation database version?

    No. VariantStudio v2.2 is programmed to use annotations from the version of the Illumina Annotation Service that was updated for VariantStudio v2.2.

  • What methods does does Illumina recommend for quantifying ChIP DNA starting material for TruSeq ChIP sample prep?

    Illumina recommends using fluorometric based methods for quantification, including Qubit or PicoGreen to provide accurate quantification of ChIP DNA. UV-spec based methods, such as the Nanodrop, will measure any nucleotides present in the sample including RNA, dsDNA, ssDNA, and free nucleotides which can give an inaccurate measurement of ChIP DNA.

  • What is the recommended read length for the libraries?

    The recommended read length is 2 x 76 bp. Paired-end reads are required for fusion detection.

  • What kind of support is provided for ChIP-Seq?

    We provide the same support as for standard genomic DNA resequencing. However, we do not provide support on antibodies or the ChIP portion.


  • What type of information should I provide when contacting Technical Support?

    For the fastest and most efficient support, supply your name, institution, contact information, and the serial number from the label on the back of your instrument. The warranty, instrument, and previous case history are associated with the instrument serial number, so this information enables Technical Support scientists to obtain the data they need to support you.

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

  • Where can I find the source code for the Isaac algorithms? Are they “open source”?

    https://github.com/sequencing. The software is released is under Illumina Open Source Software License available on https://github.com/sequencing/licenses/.The terms of the license are intended to make the software accessible to a wide variety of bioinformatics developers, computational biologists, and other users in the research community, and to encourage community development of the software. For questions about the license or its applicability, please contact Isaac-admin@illumina.com.

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

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

  • What organisms are used with ForenSeq DNA Signature Prep?

    The ForenSeq DNA Signature Prep Kit is for use with human DNA samples only.

  • Can I run more than the recommended number of samples per run on the TruSight Myeloid assay?
    Yes, but you will see lower coverage depth per sample as a result.
  • Is it possible to load a custom genome in DesignStudio for Nextera Rapid Capture Custom Enrichment?
    No it is not.
  • Where is the status.htm file?
    This report is not created by the MiSeq. However, similar information can be viewed using Sequencing Analysis Viewer (SAV).
  • What command line changes are required in CASAVA for processing the dual-index reads?

    In the demultiplexing workflow (configureBcltoFastq.pl), the --use-bases-mask parameter will need to be included if dual indexing is indicated in the sample sheet: Example: --use-bases-mask=Y*,I*,I* (for single-end runs) or --use-bases-mask=Y*,I*,I*,Y* (for paired-end runs).

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

  • If I am doing low levels of pooling with TruSeq library prep kits, what considerations do I need to make for the index selections?

    TruSeq kits support many low plex pooling options across the entire plate. Some of these combinations are outlined in the Pooling Guidelines section of the appropriate TruSeq 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

  • What washes are recommended for the MiSeq?
    All MiSeq runs include an automated wash, which rinses out the primary fluidics line, and does not require user intervention. However, the automated wash does not replace the need for a post-run wash, which should be performed after every run to flush any remaining reagents from the fluidics lines and sippers, and prevent salt accumulation, crystallization, and cross-contamination from the previous run. A maintenance wash should be performed every 30 days.
  • Can I compare expression data from different TruSeq Targeted RNA Expression runs?

    Yes, if the data from different runs is normalized with same normalizer genes. The raw data from different runs cannot be merged together into a single dataset. For more information, see the MiSeq Reporter User Guide.

  • Can I use a home-made agarose gel instead of an E-Gel for TruSeq Synthetic Long-Read DNA library prep?
    The protocol was optimized with E-Gels. Typical agarose gel dyes (stain) interfere with size selection and downstream PCR.
  • How should the Resuspension Buffer (RSB) be stored?

    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.

  • How long does it take to complete a 600-cycle run on the MiSeq?

    A 600-cycle run takes approximately 55 hours.

  • What are the storage requirements using HCS v1.4 and Flow Cell v3?

    Storage requirements for raw data are approximately 60% greater than current runs based on additional swath data and increased cluster density.

  • When is it better to use Nextera Rapid Capture Custom Enrichment for my custom target projects?

    A primary determinant is the total size of the target regions of interest. In general, smaller custom designs (up to ~0.5 Mb) are optimal for TruSeq Custom Amplicon based approaches and are best suited for sequencing on the MiSeq or NextSeq system. Nextera Rapid Capture Custom Enrichment is a better match for mid to large size custom designs (~0.5–15 Mb) and pairs best with a HiSeq, NextSeq, or MiSeq system.

  • Why do I have reads that end in adapter sequences in my small RNA sequencing results?

    Reads longer than the small RNA molecule will sequence into the adapter. For this reason, we recommend sequencing for only enough cycles to cover the small RNAs of interest, or trimming the later cycles before aligning. The sequence that would need to be trimmed from the 3' end is given here for reference.

    TGGAATTCTCGGGTGCCAAGGAACTCCAGTCACnnnnnnATCTCGTATGCCGTCTTCTGCTTG

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

  • Are HiSeq/HiScanSQ flow cells compatible with the Cluster Station?
    No. The HiSeq flow cell, also used on the HiScanSQ, must be clustered on a cBot equipped with an adapter plate suitable for the larger format of the flow cell.
  • Do I need a specific version of the bcl2fastq software package?

    Yes. To convert zipped .bcl files, use bcl2fastq v1.8.4. This version can also convert non-zipped .bcl files.

  • Are UTRs targeted?

    Yes, this kit targets 160 bp of the 5' and 3' UTR of every targeted gene. This ensures that the full gene of every targeted gene is covered.

  • Is quantitation performed before or after pooling and gel purification of the TruSeq Small RNA libraries?

    Samples can be quantified prior to pooling, but this process is often inaccurate.  At high plexity, it may be more effective to pool using equal small volumes prior to gel purification then purify,  amplify and quantitate the pooled library.  Once the pool is sequenced, any samples that did not give sufficient coverage can be re-pooled and sequenced.

  • What sequencing read length is supported on the HiSeq 3000?

    Supported read lengths for the HiSeq 3000 are up to 2 x 150 bp. Three kit sizes (50-cycle, 150-cycle, and 300-cycle) are available to support a range of read lengths.

  • What is this kit?

    This kit is a fast and easy  multiplexed amplicon assay optimized for the MiSeq System. It allows for the sequencing of 16–1536 custom amplicons (small regions of interest) across up to 96 samples. The assay also includes on-instrument variant calling for both germline and somatic variation with the MiSeq System.

  • How do I choose whether to use DNA Primer Mix A or DNA Primer Mix B for ForenSeq DNA Signature Prep?

    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.

  • Does Illumina provide classified variants?

    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.

  • How many SNPs are targeted?

    265 SNPs

  • Can I sequence and scan arrays simultaneously on the NextSeq 550 system?

    No, the BeadChip and flow cell fit in the same imaging compartment stage and use the same cameras.

  • Is the new HCS compatible with HiScanSQ?

    No. The HiScanSQ runs HCS 1.5.15 and is not upgradeable.

  • Can I modify the classification categories in BaseSpace Variant Interpreter?

    No. The software uses the following default classification schemes for tumor and germline samples:

    • Oncology analysis: FDA guidance available, general guidance available, inclusion criteria for clinical trial, and other reportable variant.
    • Germline analysis: Pathogenic, Likely Pathogenic, Variant of Unknown Significance (VUS), Likely Benign, and Benign. These categories follow ACMG guidelines.
  • What sequencing primers are compatible?

    All sequencing primers included in all TruSeq cluster kits are compatible.

  • What is the throughput of the HiSeq 3000 system?

    Each system can generate up to 750 Gb in 3.5 days with greater than 75% of bases above Q30 from a 2 x 150 bp run. This throughput enables up to 6 genomes at 30x per run per system. For more information, see HiSeq 3000/4000 System Specifications.

  • Is TruSeq RNA Sample Prep compatible with the Directional mRNA-Seq Sample Prep?

    The Directional mRNA-Seq Sample Preparation Guide provides instructions to use the Illumina Small RNA adapters, which are specific to the 3' and 5' fragment end, to generate a stranded library with the original Illumina mRNA-Seq kit. The protocol has not been optimized for the TruSeq RNA Sample Prep Kit and Illumina does not recommend this use of the TruSeq RNA Sample Prep Kit.

  • Can I generate larger fragment sizes with TruSeq RNA sample prep protocols?

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

  • How many bases constitute a region under a peak?
    See the GenomeStudio ChIP Sequencing Module User Guide available in the GenomeStudio Portal for information.
  • What is the optimal cluster density on the HiSeq?

    The recommended maximum cluster density is 750,000-850,000 clusters/mm² when using Illumina's v3 cluster generation and sequencing reagents in combination with HCS v1.4.

  • What are the requirements for the WGS 30x coverage VCF file for TruSeq Synthetic Long-Read DNA phasing analysis input?
    See the TruSeq Phasing Analysis App User Guide for a list of requirements.
  • How are the TruSight RNA Pan-Cancer and TruSight RNA Fusion workflows different?

    The workflows are identical.

  • Is quantification and normalization optional on the NeoPrep System?

    Yes, you can select 3 types of workflows on NeoPrep:

    • library prep only
    • library prep and quantification
    • library prep, quantification, and normalization

    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.

  • What is the difference in design between the TruSeq Amplicon - Cancer Panel and a TruSeq Custom Amplicon panel?

    The cancer panel is offered as an off-the-shelf standard, or fixed panel that cannot be customized. TruSeq Custom Amplicon offers the ability for users to fully customize targets of interest using DesignStudio.

  • Can the Globin-Zero Gold kit deplete degraded samples?

    Globin-Zero Gold kits remove rRNA from partially degraded samples. However, probes cannot hybridize to severely degraded samples and are therefore not effective.

  • How long does it take to switch between modes on my HiSeq system?

    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.

  • How does 3+1 quality binning work, and how does it compare to 7+1 without binning?

    The 3+1 quality binning permits a reduction in the data file sizes generated by the NovaSeq System. The mapped Q-scores for each bin are Q12, Q23, and Q37. Testing has shown highly comparable performance in HWGS between NovaSeq 3+1 qualities and HiSeq X 7+1 qualities. Although third-party analysis solutions have shown comparable performance, Illumina recommends recalibrating any settings used.

  • Are designs equivalent between TruSeq Custom Enrichment, Nextera Custom Enrichment, and Nextera Rapid Capture Custom Enrichment projects?
    Probe design algorithms have been optimized in DesignStudio for Nextera Rapid Capture Enrichment for better coverage across target regions, leading to fewer design gaps. Additional target probe spacing options have also been added for Nextera Rapid Capture Enrichment designed content.
  • What is the typical library size distribution for my TruSeq Exome libraries?

    TruSeq Enrichment Exome libraries show a tight peak of 200-400 bp. The following graph shows an example of a post-enrichment (1μl enriched 12 plex library, Bioanalyzer HS DNA chip12-plex enrichment) library distribution:

  • What level of multiplexing is supported on the HiSeq with Nextera?
    There are 96 available barcodes using 12x8 indices, combining 12 Index 1(i7) with 8 Index 2 (i5) indices.
  • Is MiSeq scalable?

    MiSeq offers scalable throughput based on read length. Illumina continues to increase read lengths, imaging area, and cluster density with improved detection and resolution. For example, at launch the MiSeq performed up to 150-cycle paired-end runs (2 x 150 bp) with greater than five million reads passing filter. Currently, the MiSeq can perform up to 250-cycle paired-end runs (2 x 250 bp)  that generates 15 million reads passing filter. For more information, see the MiSeq Product Information Sheet.

  • What is the minimum number of samples that I can prepare using this protocol?

    You can prepare 1 sample at a time.

  • I tried to cluster a set of SNPs, but no clusters are visible in the SNP Graph. What went wrong?

    SNPs are only clustered for samples selected in the Samples Table (marked in blue). If no samples are selected in the Samples Table, SNPs are clustered for all samples in the Samples Table (except non-excluded samples). Thus, if you wish to cluster SNPs for all your non-excluded samples please make sure that no samples are selected in the Samples Table at the time of clustering (e.g. by clicking onto an area in the SNP graph).

  • How many samples can be processed per TruSeq Small RNA Library Prep Kit?

    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.

  • What read length is recommended for sequencing?

    A 2×150 bp paired-end read is recommended on the MiSeq instrument for 150-250bp amplicon sizes. A 2x250 bp paired-end run is recommended for 425bp amplicon sizes.

  • What is the smallest batch size that can be prepared with this kit?

    The smallest supported batch size is 16 samples (15 samples plus one control). The 16 sample minimum batch size accounts for up to 6 freeze/thaw events of the 96 sample kit.

  • What are the compatibility requirements for using supported library prep kits on the HiSeq X system?

    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.

    Library Type

    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

  • How long does it take to perform an instrument wash on the NextSeq system?

    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.

    • Quick Wash—Uses a Tween 20 solution and takes about 20 minutes.
    • Manual Post-Run Wash—Uses a Tween 20 solution with diluted NaOCl and takes about 90 minutes.

    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.

    • If a wash was not performed, the manual wash takes about 90 minutes.
    • If an automatic post-run wash was performed, the manual wash takes 20 minutes.
  • Are TruSeq Targeted RNA Expression libraries compatible with MiSeq v3 reagents?
    Yes, they are. Note that Targeted RNA Expression libraries require 50 cycles of sequencing (single read), plus dual indices.
  • Is the ACD1 control required for the TruSight DNA Amplicon assay?
    The ACD1 control is included to allow customers to assess the success of the library preparation. Illumina recommends that customers who are new to the assay include the ACD1 control. Technical Support will not be able to troubleshoot without this control. If the ACD1 control is included in the sequencing run, it should replace a test sample. For example, if the assay recommends eight samples per run and the ACD1 control is included, then there will be seven test samples plus the ACD1 control.
  • Does Illumina offer kits containing only the oligos?

    Oligo-only kits are not available for this library prep.

  • What is the difference between the TruSeq DNA and TruSeq DNA PCR-Free protocol?
    The TruSeq DNA PCR-Free protocol does not have gel size selection or PCR amplification. It utilizes a bead-based size selection in place of the gel. The TruSeq DNA PCR-Free protocol takes about 1 day to complete in comparison to the standard TruSeq DNA protocol which takes 2 days to complete.
  • How much template does the MiSeq use?
    The MiSeq uses a total of 500 µl of denatured and diluted template for priming and clustering. However, 600 µl of template is recommended to avoid air bubbles and accommodate for instrument sipper depth. This volume does not vary for different workflows.
  • Is this assay automatable?

    This assay is being automated by some of our automation providers

    If you are interested in automating this workflow, contact your account manager.

  • Do I need a network connection for the NeoPrep System?

    In general no, because data from a run can be saved locally. However, the following operations require an internet connection:

    • Connection to BaseSpace
    • Receive and install software updates from the NeoPrep Control Software  interface.
    • Save run data from the instrument to a network drive.
    • Send instrument information to Illumina.
  • Are AMPure XP beads supplied with the kit?

    All beads required for the protocol are supplied in the kit.

  • How often do I need to wash my HiSeq system?

    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.

  • How do I choose index combinations if I only pool a small number of samples with TruSeq sample prep?

    Illumina uses a green laser to sequence G/T bases and a red laser to sequence A/C bases. At each cycle at least one of two nucleotides for each color channel need to be read to ensure proper registration. It is important to maintain color balance for each base of the index read being sequenced, otherwise index read sequencing could fail due to registration failure. For pooling strategies for a small number of samples, please refer to the "Adapter Tube Pooling Guidelines" section in sample prep guide for the kit you are using . Additionally it is recommended to create a sample sheet in the Illumina Experiment Manager (IEM) prior to performing sample prep in order to confirm appropriate index combinations not listed.

  • Why is 55 million reads recommended?

    55M reads is recommended to maximize the number of CpGs covered > 10x and to give a mean coverage of > 40x.

  • Do I need to specify a genome folder in my sample sheet for the metagenomics workflow?

    No reference genome is necessary for the MiSeq metagenomics workflow.

  • What is the necessary length of input material (i.e., if starting from DNA that is already fragmented, amplicons, etc)?
    Illumina recommends >300bp to ensure even coverage across the length of the DNA fragment. An expected drop off in sequencing coverage about 50bp from each distal end of a fragment may be seen. This is because the tagmentation reaction cannot add an adapter right at the distal end of a fragment. For PCR amplicon sequencing this can be easily averted by simply designing your amplicons to be ~100 bases larger than the desired insert to be sequenced.
  • How long does it take to perform one full cycle on the MiSeq?
    One full cycle takes approximately 6 minutes and consists of a chemistry cycle and an imaging cycle.
  • What is the typical library size distribution for my Nextera Rapid Capture Enrichment libraries?

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

    NexteraRapidCaptureLibraryDistribution

  • The kit does not include inline controls. What positive control can be used?

    Illumina recommends using 1 or more of the following recommended positive control samples. These positive control samples can be used for methylation status control.  

    Normal samples: HCC1187 normal (BL) (ATCC, catalog # CRL2323-D), NA12878 (Coriell Institute, catalog # NA12878)

    Cancer samples: HCC1187 breast cancer tumor (ATCC, catalog # CRL2322), HeLA (Biochain, catalog # D1255811), Jurkat (Biochain, catalog # D1255815)

  • Can I use strip tubes without barcode labels on the cBot 2?

    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.

  • How many tiles are imaged on a NextSeq flow cell?

    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.

    • Lanes on the high-output flow cell are imaged top and bottom in 3 swaths, or columns, for a total of 864 tiles.
    • Lanes on the mid-output flow cell are imaged top and bottom in 1 swath for a total of 288 tiles.

    For more information, see the NextSeq 500 System Guide or NextSeq 550 System Guide.

  • What is bcl2fastq2?

    The bcl2fastq2 Conversion Software converts base call (BCL) files generated on the NextSeq system. If you plan to use a third-party data analysis solution outside of BaseSpace, configure the system to operate in standalone mode and use the bcl2fastq2 Conversion Software. For more information, see bcl2fastq Conversion Software.

    Only version 2.0, or later, is compatible with NextSeq data.

  • Is the indexing primer for the paired-end flow cell compatible with the single-read flow cell?

    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).

  • What ports, domains, and encryption does BaseSpace use?

    Contact your local IT administrator if local security policies have to be modified to allow access to BaseSpace. BaseSpace uses SSL/https port 443 and the domains *.basespace.illumina.com and *.s3.amazonaws.com. Data streaming to BaseSpace is encrypted using the AES256 standard and uses SSL for protection. More information on encryption can be found at http://blog.basespace.illumina.com/2011/12/13/basespace-security/

  • My sample shows multiple fusion calls. How do I confirm a fusion call?

    To confirm fusion calls, perform additional investigation or independent validation. 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 using independent molecular biology approaches is recommended.

  • How do the TruSeq Stranded mRNA and Total RNA protocols generate stranded cDNA?
    Strand specificity is achieved by replacing dTTP with dUTP in the Second Strand Marking Mix (SMM). The incorporation of dUTP in second strand synthesis effectively quenches the second strand during amplification, since the polymerase used in the assay will not incorporate past this nucleotide. Further specificity is achieved by addition of Actinomycin D to the First Strand Master Mix Act D (FSA). Actinomycin prevents spurious DNA dependent synthesis during first strand synthesis, while allowing RNA dependent synthesis.
  • Are there any analysis workflow changes for TruSeq Nano DNA libraries in comparison to TruSeq DNA libraries?

    The TruSeq Nano DNA Library Prep Kit uses the same workflow as TruSeq DNA Sample Prep Kit. No changes have been made in analysis.

  • What consumables and equipment do I need to prepare TruSeq Synthetic Long-Read DNA libraries in my lab?

    A full list of user supplied items can be found in the Consumables and Equipment section of the TruSeq Synthetic Long-Read DNA Library Prep Guide or TruSeq Synthetic Long-Read DNA Library Prep consumables and equipment excerpt. Comparable performance is not guaranteed when using alternate equipment.

  • Is there sample data I can view?

    MiSeq datasets are available on the BaseSpace Sequencing Hub Public Data page.  For MiniSeq datasets, contact your account manager.

  • Are data sets available?

    Yes, there are examples of data from HiSeq sequencing runs in BaseSpace.

  • Are there low plexity index guidelines for this kit?

    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:

    • MiSeq when running MiSeq Control Software 2.1 or earlier
    • HiSeq when running HiSeq Control Software 2.0.12 or earlier
    • NextSeq
  • What coverage will I get with TruSeq Targeted RNA Expression?
    The amount of coverage is dependent on the level of expression of your targets in the samples you are analyzing. TruSeq Targeted RNA Expression assays target specific regions of each transcript and the level of expression depends on whether the region targeted is common between all isoforms of a gene or is transcript specific. Therefore, the amount of coverage obtained for each target is highly variable.
  • What version USB is on the MiSeq?
    The MiSeq is equipped with USB 2.0.
  • What are recurrent fusions?

    Recurrent fusions are fusions identified in the Mitelman database as present in 2 or more cases with the same morphology and topography.

  • What level of sample plexity is supported?

    The number of samples pooled pre-enrichment depends on the kit being used. 

    Kit

    Enrichment
    Reactions

    Plexity

    Catalog
    Number

    TruSeq Methyl Capture EPIC - LT

    3

    4

    FC-151-1002

    TruSeq Methyl Capture EPIC - HT

    12

    4

    FC-150-1003

    For more information, see the TruSeq Methyl Capture EPIC Library Prep Reference Guide.

  • Which method of quality assessment of RNA samples is recommended before starting the protocol?

    Quantify your library using an Advanced Analytical Fragment Analyzer. For more information, see the Evaluating RNA Quality from FFPE Samples tech note.

  • Can samples generated with the TruSeq Stranded Total RNA Sample Prep Kits with Ribo-Zero Globin or Plant be multiplexed in the same lane as human TruSeq RNA samples?
    Yes, but the indices are the same. Make sure that all samples have different index combinations.
  • What type of data files are output from TruSeq RNA Access library data analysis?
    There are various data files and plots. For more information, see BaseSpace Core Apps for RNA Sequencing.
  • What are safe stopping points in the TruSeq RNA Access Library Prep protocol and for how long?

    There are eight safe stopping points in the TruSeq RNA Access Library Prep protocol:

    1. After Synthesize Second Strand cDNA at -25°C to -15°C for up to 7 days
    2. After Ligate Adapters at -25°C to -15°C for up to 7 days
    3. After First PCR Amplification at -25°C to -15°C for up to 7 days
    4. After Library Validation at -25°C to -15°C for up to 7 days
    5. After First Capture at -25°C to -15°C for up to 7 days
    6. After Capture Sample Clean Up at -25°C to -15°C for up to 7 days
    7. After Second PCR Amplification at 2° to 8°C for up to 2 days
    8. After Second PCR Clean Up at -25°C to -15°C for up to 7 days
    For more information, see the TruSeq RNA Access Library Prep Guide.
  • How reproducible is the data?

    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).

  • Is quantitation performed before or after pooling of the libraries during TruSeq DNA PCR-Free Library Prep?

    Quantification is performed before pooling the libraries and must be done using qPCR. Other methods will quantify fragments that may not have adapters at both ends and will thus not generate clusters or sequence. Quantification by methods other than qPCR will be inaccurate. It is possible to quantify after pooling if all DNA samples are of similar quality, but this requires very consistent yields and should not be attempted by a new user. See the TruSeq DNA PCR-Free Library Prep Guide for details.

  • Will the TruSeq DNA PCR-Free Library Prep Kit work with degraded DNA samples?

    No, this kit requires high quality gDNA as starting material. Use of degraded DNA may result in low yields and loss of sample during bead clean-up steps.

  • What are the annotation sources?

    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.

  • How does the assay work?

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

  • What is the recommended TruSight One library loading concentration to reach the recommended cluster density, read depth, and run quality metrics?

    When quantifying the post-enriched library using a fluorometric method, clustering at 12.5 pM generates cluster densities ranging from 1,200 k - 1,400 k clusters/mm2 using the MiSeq v3 software and reagents. Results vary based on your method of quantification and instrument-to-instrument variability. Illumina recommends that you determine the library concentration to cluster density relationship based on your lab instrumentation.

  • What is the RNA Fusion Reference Genome?

    The RNA Fusion Reference Genome is a compacted human reference genome to enable novel fusion calling.  The RNA Fusion Reference Genome contains all of the exons in the Human Genome (according to RefSeq) with the introns removed.  It is based off of the hg19 genome.  

  • Are TruSeq DNA adapters methylated?

    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.

  • Are TruSeq DNA adapters methylated?

    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.

  • Can I use a sample sheet with the NeoPrep System?

    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.

  • What is the shelf life of the MiSeq Reagent Kit?
    Illumina guarantees that all reagent products will ship with a minimum viable shelf-life of three months.
  • Can I run GenomeStudio software on a VMware virtual machine?
    Yes. However, this is not officially supported by Illumina due to very slow performance.
  • Can the MiSeq generate FASTQ files?

    FASTQ files are generated during secondary analysis by MiSeq Reporter for most analysis workflows. To generate only FASTQ files, specify the GenerateFASTQ workflow in the sample sheet, which generates FASTQ files and then exits secondary analysis.

  • What quantitation methods does Illumina recommend for final library quantitation?

    Illumina recommends quantifying your TruSeq ChIP libraries using qPCR according to the Illumina Sequencing Library qPCR Quantification Guide.

  • Can other thermal cyclers be used with the TruSeq Nano DNA Library Prep protocol?

    Coverage of GC regions can be impacted by the model, settings, and performance of the thermal cycler used. Illumina has validated the Bio-Rad DNA Engine Tetrad 2, the Bio-Rad S1000, and the MJ Research PTC-225 DNA Engine Tetrad thermal cyclers. Other thermal cyclers may differ in their performance across the genome.

  • How should I measure the final mRNA-Seq library?
    We recommend using the Agilent 2100 Bioanalyzer to estimate final product concentration and check the size of the library. We do not recommend using only NanoDrop because its concentration estimate includes primers and other small nucleic acids.
  • How long can flow cells be stored?

    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.

  • Is BaseSpace Variant Interpreter available as a command-line tool?

    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.

  • How long does it take to generate clusters on a flow cell?

    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.

  • How are ChIP-Seq runs analyzed with Illumina software?

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

  • Can I install Windows 7 updates on the MiSeq?
    Updates to Windows are disabled. Illumina cannot guarantee system performance with future Windows 7 updates.
  • Does MiSeq require an uninterruptible power supply (UPS)?

    The use of a UPS is optional. However, a UPS is highly recommended to protect the instrument in the case of a power surge. For more information, see the MiSeq System Site Prep Guide.

  • What is the purpose of the human sequencing control in ForenSeq DNA Signature Prep?

    The human sequencing control acts to make sure that the sequencing run is performing as expected. If there is an issue with the run, the human sequencing control provides information as to whether the problem is related to the instrument or library. The human sequencing controlalso acts to allow the run to complete successfully, where degraded or low quality libraries do not have the long STR sequences required to last through 351 cycles.

  • What are the safe stopping points in this protocol?

    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

  • How many samples can be sequenced at a time?

    This kit has 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.

  • What do I need to order?
    Nextera Enrichment Kits are provided as a bundle with reagents for sample prep, enrichment and the indexes included together under a single catalog part number. To order a Nextera Custom Enrichment kit, DesignStudio is an online design tool that supports custom design and ordering for Nextera Custom Enrichment projects. Please go to http://designstudio.illumina.com/ for more information or to design a project; note that a MyIllumina account is required to access DesignStudio.
  • Which suspension buffer should I dissolve or dilute my DNA in?

    Dilute starting material in RNase/DNase-free water or 10 mM Tris-HCl, pH 8.5. Avoid EDTA containing buffers, such as TE or AE.

  • What are the lab temperature requirements for the MiSeq?

    You should maintain a lab temperature of 22°C ±3°C.

  • What is the support policy for VariantStudio?

    Our policy for the VariantStudio software is to support the previous software version for 12 months after a new version is released.  The content of the Annotation Database is aggregated from a broad range of external sources, including public and private sources.  At the time of a release for the Annotation Database, the content is locked down and versioned.  By the nature of private annotation sources, our ability to maintain annotation content for the 12-month period may not be possible in rare occurrences.  In these rare occurrences, we make our best efforts to communicate the changes to you as soon as possible to reduce any potential impact on your pipeline processes.

  • Can TruSeq Synthetic Long-Read DNA size-selected samples be stored frozen?
    No, they can be stored at 2°C to 8°C for up to 3 months. Freezing long fragmented DNA can lead to significant strand breakage leading to poor results, such as low or no yield, and short N50.
  • What read lengths are recommended for sequencing Nextera Custom Enrichment libraries?
    Based on performance data and range of library sizes, we recommend 2×36bp to 2×50bp read lengths for runs on the GA or HiSeq. For MiSeq runs, 2x150bp runs are recommended.
  • How much input genomic DNA is required for the TruSeq Exome workflow?

    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.

  • What is the recommended number of samples to pool together to sequence on the MiSeq?

    For a MiSeq 2x150 cycle run, we recommend that up to 42 samples per MiSeq run are pooled for > 1000x mean coverage per amplicon per sample in runs with 15M clusters passing filter.

  • What variants are included in the output predefined report?

    The following genes are included. The genes are also listed on the product page.

    Gene

    Target or Region

    Potential Disease States

    AKT1

    E17K

    Breast

    BRAF

    V600E/K/R/M/D/G

    Melanoma, Colon, Lung

    EGFR

    Exon 19 and  Exon 20-insertions, deletions,
    and indels  G719A/C/S, L858R, L861Q,
    S7681, T790M

    Lung

    ERBB2

    p.E770_A771insAYVM

    Breast, Lung

    FOXL2

    C134W

    Ovary

    GNA11

    Q209L

    Melanoma

    GNAQ

    Q209L

    Melanoma

    KIT

    Exons 9, 11, 13, 14, 17

    Gastric, Melanoma

    KRAS

    Codons 12, 13, 19, 59, 61, 117, 146

    Colon, Gastric, Lung

    MET

    N/A

    Lung, Colon, Gastric

    NRAS

    Codons 12, 13, 59, 61, 117, 146

    Colon

    PDGFRA

    Exons 12, 14, 18

    Gastric, Melanoma

    PIK3CA

    Exons 9, 20

    Lung, Breast, Prostate

    RET

    M918T

    Lung

    TP53

    Full CDS

    Lung, Melanoma, Ovary, Colon

  • Can custom primers be used on the HiSeq 3000?

    Custom primers have not been tested for use on the HiSeq 3000.

  • How do I access BaseSpace Variant Interpreter through my company firewall?

    BaseSpace Variant Interpreter is a web-based application. To make sure that you can access it through your company firewall, open port 443.

  • What applications are supported on the HiSeq X system?

    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.

  • Can the sample sheet for the MiSeq FGx run be created using the Illumina Experiment Manager (IEM)?

    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.

  • What is the delivered volume for a maintenance wash?

    Positions

    Delivered Volume

    8 SBS positions

    74 ml

    10 paired-end positions

    52 ml

    All positions

    15.75 ml per lane

  • Where are data stored after they are imported into VariantStudio?

    The desktop version of Illumina VariantStudio stores data locally in the VariantStudio project file. During the import of VCF files, data are loaded to the desktop memory. To save the data, designate a location to save the project, which can be reopened later.

  • How should ChIP DNA be quantitated?

    We recommend quantitating ChIP DNA with the most sensitive method available, taking into account the low amount of DNA that is often available. Fluorescent dye-binding assays such as PicoGreen or Qubit offer high accuracy, assuming precise pipetting. The Bioanalyzer and High Sensitivity chips can also give good quantitation, and allow you to check the size and purity of your sample.

  • How do I quantify TruSeq DNA PCR-Free libraries?

    The TruSeq DNA PCR-Free protocol does not contain a PCR enrichment step to select for completed ligation products, therefore quantitation must be done by qPCR as other methods will quantify fragments that may not have adapters at both ends and will thus not generate clusters or sequence. See the Consumables and Equipment section of the TruSeq DNA PCR-Free 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.

  • What applications can I run on the MiniSeq System?

    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.

  • What enrichment levels can I expect when using the TruSight One kit?
    The expected final percentage of data from enriched regions is 50-70%, but vary across enrichments and can be influenced by the quality of starting material.  The enrichment wash steps are key to reducing non-specific DNA binding and require samples to be maintained at the indicated temperature.  Too low or too high temperatures can result in lower percent enrichments and decreased yields.  Make sure that the proper instrumentation is used and that the streptavidin beads are properly resuspended.  Key points for user-supplied lab equipment, equipment temperatures settings, and enrichment wash procedures are outlined in the TruSight One User Guide.
  • What should I do if I get an error indicating that the program can't be found? For example: -bash-3.1$ GAPipeline-1.3.2/bin/illumina2srf -o lane_1.srf / /Data/IPAR_1.3/Bustard1.3.2_01-03-2009/s_1_*_qseq.txt -bash: /GAPipeline-1.3.2/bin/illumina2srf: No such file or directory
    If you get this message, you should explicitly install the io_lib. To do this, run the following command from the Pipeline Install directory: /GAPipeline_1.3.2 make WITH_IO_LIB=1 install, then retry the command from Q2, above.
  • Is there a clear minimum coverage (%) that an alternate allele has to reach to be called a heterozygous (minimum minor allele frequency of 20 or 30%)?
    The Isaac variant caller is based on a Bayesian model and will assign probabilities to different possible variant calls so no specific minimum exists.
  • What molecules are removed with the TruSeq Stranded Total RNA Sample Prep Kit with Ribo-Zero Globin?
    The Ribo-Zero removal mix targets cytoplasmic rRNA, mitochondrial rRNA, plus globin mRNA.
  • Can I change settings on the RNA Fusion module?

    Yes, you can change 3 settings:

    Minimum Breakpoint Distance - Excludes genes that are in close proximity and may be read through events rather than fusions.  The default distance was determined empirically from our test dataset. This was a safeguard to prevent calling read-through transcripts as fusions.  The minimum breakpoint distance is the shortest possible distance between the genes, using either the start or end coordinate. It is not determined by the breakpoint position.

    •     Too close = likely false positives
    •     Too far = may miss real fusions

    Confidence Score Filter - Set by Illumina based on internal testing. The scores are calculated as a weighted average of individual features (for example, split reads, fusion contig alignment length, etc).  If the fusion does not meet the confidence score, it is not shown, helping to eliminate false positives.  For detailed information about scoring calculations and what is being evaluated as part of the confidence score, see the Local Run Manager RNA Fusion Analysis Module Workflow Guide (document # 1000000010786).

    Confidence Score Threshold - Value established by Illumina. Fusion calls with a confidence level above this threshold are considered "High Confidence."

  • What does it mean if a fusion is called in one replicate but not in another?

    It can indicate a lower confidence in the fusion call, which can be due to low expression levels. An orthogonal approach to confirm results is recommended.

  • What percent of PF reads aligned to rRNA is expected?

    The %PF reads aligned to human ribosomal RNA is typically ≤ 8%.

  • Where can I find more detailed information about my sample results?

    Follow the directory path: RNA_Fusion_Analysis/samples/[SampleName]/

    In this folder, there are 3 folders named Align, GeneCounts, and MantaFusion. These folders contain the number of read counts, their passing values, and other detailed information.

  • What is the recommended loading concentration for sequencing on the MiSeq?
  • Does the TruSight Myeloid Sequencing Panel detect partial or tandem duplicates?
    MiSeq Reporter does not detect duplication events. Reads from this type of rearrangement are not expected to align and will likely be placed in the Unaligned folder by MiSeq Reporter.
  • How long can ForenSeq DNA Signature Prep libraries be stored before being sequenced?

    Final pooled libraries can be stored at -25°C to -15°C for up to 3 months.

  • How long after completing an array scan can I start a sequencing run on the NextSeq 550 system?

    You can start a sequencing run when the NextSeq 550 system completes the array scan.

  • Why is the message "Sequence Not Found" sent after I submit SNPs chosen using ADT?
    This message means that the SNP sequence cannot be found in our internal database, which is a filtered version of dbSNP. We filter out any SNPs that are not appropriate for the Illumina platform, such as insertions/deletions, multiple nucleotide SNPs, and SNPs with ambiguous or multiple localizations.
  • What read length is recommended?

    The recommended read length is 2 x 101.

  • What quality of DNA is required and how should DNA quality be assessed?

    Commercially available or laboratory validated DNA extraction methods typically yield DNA that is compatible with this assay. DNA purity should have an A260/A280 ratio of 1.8–2.0. PicoGreen is recommended. If using FFPE DNA at the supported amplicon sizes (150 or 175bp amplicons), follow the recommended QC steps to select DNA of appropriate quality.

  • Do I need to purchase AMPure beads if using the TruSeq Nano DNA Library Prep Kit?

    No, the kit contains Sample Purification Beads (SPB) that are used for size selection and clean-up steps. They do not require the user to supply AMPure beads separately.

  • Can I use a thermal cycler in place of a microheating system in the TruSeq Targeted RNA Expression protocol?
    Yes. However, the samples must be transferred to a MIDI plate for the Wash, Extend, and Ligate Bound Oligos procedure, then transferred to a PCR (HSP) plate for the PCR Amplification procedure.
  • What is a NeoPrep library concentration when normalization is performed by the instrument?

    The complete protocol on NeoPrep results in 10 µl of 10 nM library after the library droplet is collected from the library card, diluted in RSB, and then separated from the oil using the library separation tubes.

    This is true if library prep, or library prep and quantification, or library prep, quantification, and normalization are selected during run setup.

    The NeoPrep reported quantification and normalization values take into account the library dilution in RSB.

  • What is paired-end analysis?
    Paired-end analysis sequences both ends of a DNA fragment. If the fragments are of known size, this method can facilitate de novo sequencing of repetitive elements and help to identify structural variation.
  • How does DesignStudio select probes?

    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 more information, see the DesignStudio online help.

  • What DNA input amount is required?

    The assay uses between 150 and 250ng of DNA, depending on the quality of DNA and number of targets in the CAT.

  • How do I mix the RNA sample with the magnetic beads?

    Make sure that you add the RNA sample to the washed, room-temperature magnetic beads. Do not add magnetic beads to the RNA sample.

  • What are the main protocol differences between the Nextera kit and the new Nextera XT kit?
    The Nextera XT kit introduces a faster tagmentation step and a bead-based library normalization with only 1ng of input DNA. This workflow has been optimized for the Nextera XT kit and reagents between the Nextera kit and the Nextera XT kit are not interchangeable.
  • What flow cells and reagents are supported on the HiSeq X system?

    The HiSeq X system uses patterned flow cell technology. Reagents provided in the HiSeq X Reagent Kit are designed and optimized for the HiSeq X flow cell. Non-patterned flow cells and SBS kits designed for other HiSeq models have not been validated and are not supported for use on the HiSeq X system.

  • For dual-indexed libraries, how many cycles are performed for index reads?

    Dual-indexed runs on the HiSeq comprise 8 bp of index sequence rather than 6 bp plus a seventh for phasing calculations. For more information, see the user guide for your sequencing instrument.

  • What percent strandedness is expected?

    For good quality samples, > 98.5% strandedness is expected.

  • What can I do if my tagmentation reaction has resulted in longer/shorter fragments than I expected? Will this impact my results?
    The optimal fragment length range for TruSight One libraries is ~300 bp -1 kb. A higher proportion of longer or shorter fragments can affect enrichment and/or clustering. Ensure best practices and Illumina recommended labware and consumables are used.
  • Can custom primers be used on the MiSeq?

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

  • What level of indexing or barcoding is available?

    Up to 96-plex indexing is supported for TruSeq Custom Amplicon, Nextera, Nextera XT, and TruSeq DNA Sample Prep Kits.

  • Because only a single strand is captured, is information on the opposite strand complementary to the CpG sites lost?

    Lister, Ecker, and colleagues (Nature 2009) found that 99% of CpG sites on the complementary strand share the same methylation state. Therefore, methylation of the complementary strand can be inferred from single strand methylation data in most cases. 

  • Is there a minimum number of indices I need to use?
    For dual indexing, at least 2 barcodes/indices need to be present for each Index Read. For less than 6 samples, Illumina recommends using single indexing. Please refer to the pooling guidelines in the Nextera DNA Sample Preparation Guide.
  • Does GenomeStudio software currently offer features for small RNA Sequencing and Tag Sequencing data analysis and visualization?
    Not at this time.
  • Can qPCR be used to quantitate small RNA libraries?
    Yes. For the most accurate measurements, the control library used to generate a standard curve for qPCR should be as similar as possible to the library being measured, which makes it necessary to use a small RNA library as a control library.
  • What quality of DNA is required and how should DNA quality be assessed?

    Commercially available or laboratory validated DNA extraction methods typically yield DNA that is compatible with this assay. DNA purity should have an A260/A280 ratio of 1.8–2.0.

     

  • How many boxes come in a kit?

    For both the LT and HT configurations, 1 kit contains 5 boxes.

  • What’s new in NextSeq Control Software (NCS) v1.4?

    NCS v1.4 enables the use of the NextSeq 500/550 Kit v2, supports BaseSpace run monitoring in standalone mode, introduces a reagent purge feature, can proceed to sequencing automatically after a successful pre-run check, and includes a Quick Wash option. 

  • What software does Illumina offer for analysis of TruSeq RNA Access libraries?
    Illumina offers the BaseSpace Core Apps for RNA. Learn more about BaseSpace.
  • Do I need an upgraded MiSeq to run high-plexity samples?

    You do not need an upgraded MiSeq to run these samples. However, consider the coverage needed for your study when designing an experiment. An online calculator is provided in DesignStudio to help with these calculations.

  • Can the TruSeq Amplicon - Cancer panel be sequenced on a HiSeq?
    The TruSeq Amplicon - Cancer panel is fully supported on the MiSeq. This panel takes advantage of the long read lengths (2 x 150 bp) and the integrated software solution built in to the MiSeq. TruSeq Custom Amplicon and the TruSeq Amplicon – Cancer Panel will be compatible with the HiSeq2500.
  • When should you use the TruSight RNA Pan-Cancer Panel and when should you use TruSeq Targeted RNA Expression?

    TruSight RNA Pan-Cancer is a large gene panel covering 1385 cancer-related genes. TruSeq Targeted RNA Expression typically provides smaller panels and fusion detection is more difficult. In addition, TruSight RNA Pan-Cancer has discovery power for fusion detection because only 1 gene fusion partner is required. With TruSeq Targeted RNA Expression, oligos must be designed either side of a known breakpoint. TruSight RNA Pan-Cancer is more amenable to low input FFPE.

  • What is the recommended RNA input amount? What is the recommended input amount for FFPE RNA?

    The Protocol is optimized for 10-100ng of human total RNA. Lower input amounts may result in low yield and reduced sensitivity. Use 10ng for high-quality universal human reference total RNA as input. 

    For 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. 

    Quality

    DV200

    Input Requirement Per Reaction

    High

    > 70%

    20 ng

    Medium

    50-70%

    20-50 ng

    Low

    30-50%

    50-100 ng

    Too degraded

    < 30%

    Not recommended

    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.

    Rarely, low quality samples can meet the DV200 ≥ 30% spec, but may still result in false negatives.  These samples tend to have high duplicate sequencing reads and/or low percent unique reads.

  • What Nextera Rapid Capture Enrichment Kits are available and how many samples can be processed with each kit?

    Kit Name

    Catalog #

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

    FC-140-1000

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

    FC-140-1083

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

    FC-140-1086

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

    FC-140-1089

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

    FC-140-1001

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

    FC-140-1002

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

    FC-140-1003

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

    FC-140-1004

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

    FC-140-1005

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

    FC-140-1006

    Nextera Rapid Capture Custom Enrichment (48 Samples)

    FC-140-1007

    Nextera Rapid Capture Custom Enrichment  (96 Samples)

    FC-140-1008

    Nextera Rapid Capture Custom Enrichment (288 Samples)

    FC-140-1009

  • What actual assay performance can I expect from my design?

    DesignStudio returns high confidence amplicon designs that have delivered unprecedented amplicon multiplexing performance. You can expect to see specificity and uniformity > 70%/80% respectively. In practice, we have observed specificity and uniformity > 90% for hundreds of designs.

  • Would there be issues demultiplexing current TruSeq libraries if a dual index run was done?
    No. You must specify the correct –use-bases-mask and use the appropriate sample sheet to enable demultiplexing. For more information, see the CASAVA User Guide for details on sample sheets and demultiplexing commands.
  • What probe pool is used in TruSeq RNA Access Library Prep?
    Coding Exome Oligos (CEX) are used, which are also used in Nextera Rapid Capture Exome Enrichment.
  • What is a TruSeq Targeted RNA Expression manifest file?
    The manifest file is a .txt file which contains the probe and target sequences for your order. It is used by MiSeq Reporter for on-instrument alignment. You must use a manifest file for the analysis of TruSeq Targeted RNA Expression data.
  • What control software version is compatible with current kits and library prep methods?

    Use HiSeq Control Software v3.3 and Real-Time Analysis v2.3, or later. Previous versions of HiSeq Control Software and Real-Time Analysis are not compatible.

  • What third party software can be used for analysis of TruSeq RNA Access libraries?
    Illumina's BaseSpace Core Apps for RNA includes the industry standard TopHat and Cufflinks analysis pipeline. Additional, third party analysis tools are also available.
  • How do I perform TruSeq RNA without using PCR to enrich DNA fragments that have adapter molecules on both ends?

    The number of PCR cycles should be minimized to avoid skewing the representation of the library.  Illumina recommends 15 cycles of PCR, which has been shown to provide the best performance in terms of coverage, reproducibility, and quantity of material.  Although not recommended by Illumina, if you want to eliminate PCR, begin with a large amount of starting material (~5 μg) to generate equivalent yield as the standard protocol.  Note that at least one cycle of PCR is required to open the forked adapters. 

  • What sequencing platforms are compatible?

    Libraries produced with this kit are compatible with all sequencing platforms that allow methylation sequencing. However, to obtain the industry standard of 55 million reads per sample, NextSeq and HiSeq are recommended.

  • How should I test the quality of RNA as the TruSeq RNA starting material?

    Illumina recommends that you check total RNA integrity following isolation using an Agilent Technologies 2100 Bioanalyzer with an RNA Integrity Number (RIN) value greater than or equal to eight.

  • How much input RNA do I need for TruSeq Targeted RNA Expression?
    The protocol is optimized for 50 ng of total RNA. For degraded samples with ≥ 200 bp fragments, Illumina recommends the RNA input be optimized for 100–200 ng, depending on the RNA quality.
  • Can I expect comparable library preparation performance between the TruSeq DNA PCR-Free LT and HT kits?
    Yes, both the LT and HT kits will give comparable results.
  • What does TruSight DNA Amplicon data look like? Is demo data available?
    For an example of data from a TruSight DNA Amplicon experiment, refer to the data set available via BaseSpace. This sample data set is from the TruSight Myeloid Sequencing Panel sequenced on the MiSeq with MiSeq v3 reagents.
  • Do I have to run two flow cells at the same time on a HiSeq 2000?
    No. You can choose to run only one flow cell at a time.
  • Are pre- and post- PCR areas required to run the Nextera Rapid Capture Enrichment protocol?
    It is advisable to separate pre- and post-PCR due to amplicon generation in the protocol. For more information, see the Best Practices section of the Nextera Rapid Capture Enrichment Guide for additional information.
  • Are FFPE samples supported?

    FFPE samples are not supported.

  • How do I use BaseSpace for run monitoring only, such as SAV functionality in BaseSpace?
    Run monitoring with BaseSpace is selected during run setup.
  • Do I need a cBot to cluster HiSeq 3000/4000 flow cells for sequencing on my HiSeq 3000?

    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.

  • How many samples can be generated per TruSeq HT library prep kit?

    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.

  • Where is BaseSpace Variant Interpreter hosted? Where is data stored?

    BaseSpace Variant Interpreter is hosted on Amazon Web Services. Storage is account-specific and is also hosted on Amazon Web Services.

  • Is a sample sheet required for TruSeq Synthetic Long-Read DNA library prep? If so, what application do I use in the Illumina Experiment Manager software?
    A sample sheet is required prior to starting a sequencing run and enables the correct identification and processing of libraries after the sequencing run is streamed to BaseSpace. Use IEM v1.8 or later and select the TruSeq Synthetic Long-Read DNA kit and the HiSeq FASTQ Only application.
  • What is the input amount required for TruSeq DNA PCR-Free Library Prep?

    1 µg of high quality gDNA is required for final libraries with an average 350 bp insert size and 2 µg of high quality gDNA is required for a 550 bp insert size.

  • How do I perform amplicon sequencing on a MiSeq?

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

  • Can the TruSeq Sample Prep protocols be run without controls?

    Yes. To run the protocol without the controls, substitute Resuspension Buffer (RSB) for the control mixture at each step. See the appropriate TruSeq DNA or RNA sample preparation guide for details.

  • What types of SBS kits are available for the HiSeq?
    Illumina currently offers two SBS kits for the HiSeq: a 200-cycle kit and a 50-cycle kit. Both kits contain the same formulations and only differ in volume.
  • How do I check the quality of my library?

    Use an Agilent Technologies 2100 Bioanalyzer to check the quality and intended size distribution of the Covaris sheared sample and the final library. For examples of Bioanalyzer traces and library size distributions, see the reference guide.

  • Why must I gel purify before the PCR step?
    To avoid amplification of adapter dimers.
  • How much input RNA is too much?

    The Ribo-Zero Epidemiology kit supports standard input of 500 ng–2.5 µg and low input of 100–500 ng total RNA samples. Do not exceed the maximum amount of RNA. Quantify the input total RNA using a Qubit Fluorometer. The RNA concentration reported by the Bioanalyzer or NanoDrop might not be accurate.

  • Which genes are targeted in this panel?

    TruSight RNA Fusion targets 507 genes involved in fusions in cancer. The gene list can be downloaded from the TruSight RNA Fusion Downloads page and the product page.

  • How much input RNA is too much?

    With the exception of the Epidemiology kit, Ribo-Zero kits support 1–5 µg total RNA for standard input or 100 ng to 1 µg total RNA for low input. Do not exceed the maximum amount of RNA. Quantify the input total RNA using a Qubit Fluorometer. The RNA concentration reported by the Bioanalyzer or NanoDrop might not be accurate.

  • How long are the oligos?

    The oligos are 80 mers?

  • How long is each Index Read?
    Both Index 1 (i7) and Index 2 (i5) are 8 bp in length.
  • How are the components different from the TruSeq RNA Access kit?

    This 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.

  • How long can maintenance wash solution be stored after initial preparation?

    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.

  • How is the Illumina Nextera kit different from the legacy Nextera kit from Epicentre?
    The Illumina Nextera kit has improved master mixes resulting in an easier protocol with less tubes. Additionally, there is an optimized PCR that does not require the purchase of any additional components and an optimized tagmentation reaction. Finally, the new dual indexing strategy allows 96 indices per lane and samples can be sequenced on all Illumina sequencing platforms.
  • Can I use more than 100 ng of total RNA as input?

    Yes, up to 500 ng of total RNA input has been tested.

  • What is the difference between Nextera Rapid Capture Exome Enrichment and TruSight Exome?

    The Nextera Rapid Capture Enrichment Exome is designed to cover > 98% of coding content of the human genome, spanning ~45 Mb of genomic content. The TruSight Exome is a focused subset of the exome targeting 7 Mb of genomic targets associated with a variety of inherited diseases.

  • Can the Ribo-Zero kit deplete degraded samples?

    Ribo-Zero kits remove rRNA from partially degraded samples. However, probes cannot hybridize to severely degraded samples and are therefore not effective.

  • What indexing options are available on the HiSeq X system?

    Although the TruSeq Nano DNA and TruSeq DNA PCR-Free library prep kits are compatible with dual indexing, only 8 bp single indexing is supported on the HiSeq X system.

  • Is it OK if I still see primers after the final Qia-Quick PCR cleanup?
    Yes, this is OK. Their presence will not impact the performance of the assay.
  • Does a HiSeq 1500 or HiSeq 2000 need to be upgraded to a HiSeq 2500 to run the HiSeq v4 chemistry?
    No. All eligible HiSeq 1500 and HiSeq 2000 systems only need the FPGA and software upgrades to use the HiSeq v4 reagents.
  • How many samples should I load per run when performing the TruSight Myeloid assay?
    Illumina recommends the following to give > 95% of amplicons at > 500x coverage:
    • 8 samples per MiSeq v3 run
    • 4–5 samples per MiSeq v2 run
    • 42 samples per NextSeq 500 Mid Output run. Customers can also run fewer samples to achieve higher coverage economically.
    • 96 samples per NextSeq 500 high output run or HiSeq 2500 rapid run. (96 samples is the maximum per run due to the number of indexes available.)
  • Does BaseSpace Variant Interpreter require an internet connection?

    Yes, the software requires an internet connection. All software operations are conducted in the cloud.

  • Will variants be called across the entire length of the sequenced amplicon?

    Variant calling is only performed in the amplicon region lying between the upstream and downstream probe locations. The probe regions are not included in variant calling. MiSeq Reporter uses the manifest file for each design to avoid calling variants in probe regions that are synthetic oligonucleotides and not biologically relevant.

  • What percent duplicates is expected?

    The range of percent duplicates is expected to be < 32% for controls (UHR) at 0.5M subsampled reads. We observed 20-80% duplicates in customer samples with most successful samples having < 60% duplicates..

     

     

     

     

     

     

     

  • Which reference genome should I use?

    Use the Homo sapiens (PAR-masked)/hg19 (RefSeq) or Homo sapiens (PAR-masked)/hg 19 (Gencode) reference genome. Results can vary depending on the reference used because of different annotations within those genomes.

  • Can I use any kit version with NCS v1.4?

    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.

  • Can I expect comparable library preparation performance between the TruSeq Nano DNA LT and HT kits?
    Yes, both the LT and HT kits will give comparable results; however, the LT kit is recommended for preparing less than 24 samples at a time.
  • What method should I use to quantify my total RNA input?

    Use NanoDrop to quantify the RNA concentration. If NanoDrop is not available, use a fluorometric method.

  • Can I use a custom Index 2 primer on the NextSeq system?

    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.

  • How long does it take to complete a sequencing run on the NextSeq system?

    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.

  • How many lanes does the MiSeq flow cell have?

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

  • Can all 96 indexes support use of demultiplexing with mismatch=1?

    Yes, the nucleotide distance of all the indices is such that mismatch of 1 still makes a unique index.

  • Can all 96 indexes support use of demultiplexing with mismatch=1?

    Yes, the nucleotide distance of all the indices is such that mismatch of 1 still makes a unique index.

  • Can TruSeq v3 reagents be used on a system that has been upgraded for HiSeq v4?
    Yes. You can continue to use TruSeq v3 reagents as well as HiSeq v4 reagents. The chemistry type is specified when you select the mode before starting the run.
  • Do I need a cBot to cluster HiSeq 3000/4000 flow cells for sequencing on my HiSeq 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.

  • What probe pool is used in the TruSight RNA Pan-Cancer Panel?

    The probes are specifically designed to target 1385 cancer genes and to detect fusions by spanning all exons of every gene.

  • What are the requirements for importing whole genome data or large gVCF files?

    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.

  • Can secondary analysis by MiSeq Reporter be delayed after a sequencing run completes?
    No, secondary analysis cannot be delayed automatically. Secondary analysis will automatically stop if you begin the run setup steps for a subsequent sequencing run on the MiSeq.
  • What quality of DNA is required for the TruSight DNA Amplicon assay and how should this be assessed?
    Commercially available or laboratory validated DNA extraction methods typically yield DNA that is compatible with this assay. DNA purity should have an A260/A280 ratio of 1.8—2.0. PicoGreen is recommended.
  • Do I need to perform indexing if preparing samples with Nextera?
    No, you can run single samples without indexing. However, you must order one of the two available Index Kits in order to perform the PCR required for the library prep: either the 24 Index Kit with sufficient reagents for 96 samples (Catalog # FC-121-1011) or the 96 Index Kit with sufficient reagents for 384 samples (Catalog # FC-121-1012). When clustering/sequencing these samples on cBot/Cluster Station and HiSeq/HiScanSQ/GAIIx, even if you do not perform indexing (single or dual), you will need to use the new TruSeq Dual Index Sequencing Primer Kits (Single Read Kit is Catalog # FC-121-1003 and the Paired End Kit is Catalog # PE-121-1003).
  • How do I check the quality of my library?

    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.

  • How can I calculate required coverage?
  • Do NextSeq reagents use the same SBS chemistry as the HiSeq and MiSeq reagents?

    All Illumina sequencers use the Illumina patented reversible-terminator SBS chemistry. The NextSeq system employs the latest evolution in SBS technology: a novel 2-channel SBS method that supports reduced cycle time and data processing time.

  • With the release of the TruSeq Nano DNA Library Prep Kit, will the TruSeq DNA Sample Prep Kit still be available for purchase?

    Illumina has discontinued TruSeq DNA Sample Prep kits. Customers are encouraged to switch to either the TruSeq DNA PCR-Free Library Prep Kit or the TruSeq Nano DNA Library Prep Kit.

  • What do the different target Desired Probe Spacing options represent, when designing probes across my Nextera Rapid Capture Custom Enrichment targeted regions?
    The Desired Probe Spacing setting allows you to adjust the way DesignStudio places enrichment probes within the region of interest. Increasing this setting allows DesignStudio to place probes more closely to one another within a given region. The target center-to-center spacing for neighboring 80 mer probes at each setting is:
    • Standard—230 bp
    • Intermediate—180 bp
    • Dense—120 bp
    • Adjacent—80 bp
    • Overlapping—60 bp
    Note that altering the Desired Probe Spacing setting allows the DesignStudio algorithms to place probes according to the spacing listed, but does not guarantee this spacing. DesignStudio does not place probes that are incompatible with other design rules just to achieve desired spacing.
  • How do I analyze TruSeq Synthetic Long-Read DNA data?
    Purchase of the kit includes access to TruSeq Long-Read Assembly or TruSeq Phasing Analysis applications in BaseSpace.
  • How much input RNA is too much?

    The Globin-Zero Gold kits support 1–5 µg total RNA for standard input or 100 ng to 1 µg total RNA for low input. Do not exceed the maximum amount of RNA. Quantify the input total RNA using a Qubit Fluorometer. The RNA concentration reported by the Bioanalyzer or NanoDrop might not be accurate.

  • What are the DNA input requirements TruSeq Synthetic Long-Read DNA Library Prep?

    Illumina recommends 50 μl input DNA at 10 ng/μl.

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

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

  • Does BaseSpace Variant Interpreter support the genome VCF (gVCF) file format?

    Yes. The software supports VCF and gVCF file formats. For more information, see the BaseSpace Variant Interpreter Beta Online Help.

  • Can I perform Single Read runs and still get both index sequences?
    Yes. Please select the appropriate workflow based on the flow cell type (Single Read or Paired End) when starting your run in HCS.
  • How can I optimize cluster formation?

    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.

  • How do I format my sample sheet?

    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.

  • Can I use SuperScript III instead of SuperScript II?
    Illumina recommends following the protocol as closely as possible. The protocol was optimized with SuperScript II.
  • Are the Bisulfite Conversion reagents supplied with the kit?

    The bisulfite conversion reagents required for the protocol is supplied in the kit from Zymo Research.

  • Are Nextera Enrichment kits supported on MiSeq?
    Samples enriched with Nextera Enrichment kits can be sequenced on MiSeq. Full bioinformatics analyses may have to be performed offline, although a MiSeq Reporter workflow for enrichment samples is expected at a future date.
  • Can index reads on TruSeq dual-indexed libraries be rehybridized?

    For paired-end flow cells, only Index 1 can be re-hybed, and this must occur before Index 2 begins. For single-read flow cells, Index 1 and Index 2 can be rehybridized before the run completes.

  • How many TruSight One reactions are included in each kit? What are the minimum and maximum numbers of samples that I can multiplex per enrichment in my TruSight One enrichment pools?

    The TruSight One 9-sample kit provides sufficient reagents to prepare a maximum of three (3) enrichment reactions of up-to 3-pooled samples per enrichment.  The TruSight One 36-sample kits provide sufficient reagents to prepare a maximum of three (3) enrichment reactions of up-to 12-pooled samples per reaction. Single sample enrichments are possible, but the reagent volumes are optimized for preparing three sets of pooled sample libraries at either the 3- or 12-plex levels mentioned previously.  

  • What additional equipment is required to run a HiSeq?
    The HiSeq requires a cBot cluster generation system, a network file system, and a mobile lab bench with locking casters (recommended).
  • How can I improve the designability and coverage in my DesignStudio Project?
    1. Increasing the size of the target to design against can rescue previously ‘undesignable’ regions. The increased size of a target gives DesignStudio a little more flexibility to fit a higher scoring amplicon over the desired target bases.
    2. Use the 'Avoid SNP’ feature – With this option, DesignStudio will be more aggressive when placing amplicons by putting less importance of the location of SNPs in the design process. This can help rescue ‘undesignable’ regions if DesignStudio avoided regions with low frequency or poorly annotated SNPs in the database, leading to low designability. DesignStudio uses dbSNP 138 or 1000 Genomes (human hg19), SNP 128 (mouse mm9), SNP 125 (rat rn4) and Ensembl UMD3.1 SNPs (bovine) to position probes that avoid SNP and indel locations to improve accuracy and performance of the design for that region.
    3. Change the context of the panel – for example, putting a highly homologous or high GC rich target sequence into the same multiplex design can be problematic for designing probes to amplify each target discretely. Moving problematic regions into a separate design can frequently improve the designability.
  • How are counts normalized in the RNA Sequencing Module?
    See the GenomeStudio RNA Sequencing Module User Guide in the GenomeStudio Portal for more information.
  • What read length is recommended for sequencing with MiSeq?

    TruSeq Amplicon - Cancer Panel requires 2 x 150 bp read length. This read length provides bidirectional sequencing of amplicons in the panel.

  • Why might a fusion not get called?

    Several factors can cause a fusion to not get called, including:
    - Low expression levels of the fusion gene. More sequencing read depth or lower fusion score threshold may be required.
    - Low quality of the sample. More sample input RNA may be required.
    - Close proximity of 2 genes in the same orientation, on the same chromosome (eg, STIL-TAL1). Reducing the default breakpoint distance thresholds in the LRM module can help identify these fusions, but may also display more false positive calls.
    - Differences in bioinformatics algorithms used.
    - Differences in reference genomes. GENCODE has higher genomic coverage than RefSeq and fusions between exons that are not annotated are not called in RefSeq. For example, an EML4 transcript has an exon that is annotated in GENCODE but not RefSeq, meaning fusions at that exon would not be called when using the RefSeq reference.

  • Can I use DEPC water instead of RNase/DNase free water?

    DEPC water is not recommended. Contaminants can inhibit parts of the reaction.

  • Does TruSeq Synthetic Long-Read DNA data analysis require BaseSpace?
    Yes, sequencing runs must be streamed to BaseSpace to utilize the corresponding TruSeq Long-Read Assembly and TruSeq Phasing Analysis apps on BaseSpace for analysis.
  • What if I have a magnet other than the one recommended by Illumina for TruSeq library prep?

    Cleanup procedures have only been optimized and validated using the magnetic stand specified in the appropriate TruSeq DNA or RNA library prep guide. Comparable performance is not guaranteed when using other magnets. Other magnets can be used, however you may want to test how long samples need to sit on the magnet, as times may vary from the protocol.

  • Is too much DNA a concern with ForenSeq DNA Signature Prep?

    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. 

  • What is an amplicon?

    An amplicon is a fragment of DNA that is produced from an amplification event such as PCR. 

  • Should the libraries be run as single-read or paired-end?

    Run the libraries as paired-end.

  • Can I use a TruSeq HT library prep kit with a TruSeq LS library prep protocol? Can I use a TruSeq LT library prep kit with a TruSeq HS library prep protocol?

    Yes, the low sample (LS) and high sample (HS) workflows may be applied with both the high throughput (HT) and low throughput (LT) kit configurations. Please refer to the user guide for the TruSeq library prep kit that you are using for Illumina's recommendations on kit and protocol combinations to ensure the best results.

  • Are there any workflow changes associated with the HiSeq X Reagent Kit v2.5?

    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.

  • What sequencing primers are needed for TruSeq v2/LT or HT kit library prep?

    For TruSeq v2/LT kits, standard sequencing primers included in the cluster generation kits are required.

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

  • Does Illumina provide classified variants in the Classification Database?

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

  • Does TruSight DNA Amplicon use the same PCR/sequencing primers as TruSeq Custom Amplicon or Nextera?
    TruSight DNA Amplicon uses the same PCR/sequencing primers as TruSeq Custom Amplicon. Nextera uses different oligos.
  • How many flow cells can I run on the HiSeq?
    The HiSeq 2000 is a dual flow cell system, which allows you to run two flow cells simultaneously. The HiSeq 1000 is a single flow cell system.
  • What flow cells and reagents are supported on the HiSeq 4000?

    Reagents and flow cells provided in the HiSeq 3000/4000 SBS Kit and the HiSeq 3000/4000 Cluster Kits are designed and optimized for the HiSeq 4000 and HiSeq 3000 systems. Reagent kits designed for other HiSeq systems are not compatible with the HiSeq 4000 and HiSeq 3000 systems.

  • Can I cluster any Illumina library type on the cBot 2?

    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.

  • What is the shelf life of the kits?

    One year from the date of manufacture. The kit contains an expiration date on the label.  Illumina guarantees at least 3 months from the date of receipt.

  • What is the minimum input material for small RNA sequencing?
    The protocol was optimized using 1.0 μg of total RNA.
  • What is the difference between DNA Primer Mix A and DNA Primer Mix B in ForenSeq DNA Signature Prep?

    DNA Primer Mix A—Contains primer pairs for 59 STRs (including 27 autosomal STRs and 8 X and 24 Y haplotype markers) and 95 identity-informative SNPs.

    DNA Primer Mix B—Contains all markers in DNA Primer Mix A, plus primer pairs for 56 ancestry-informative SNPs and 22 phenotypic-informative SNPs (two ancestry-informative SNPs are also used for phenotype prediction).

  • Can I use different sequencing primers in the 2 rapid flow cell lanes?

    No. The same sequencing primer is distributed across both lanes of a rapid flow cell as part of the HiSeq 2500 on-instrument cluster generation workflow.

  • Can BaseSpace Variant Interpreter be used in a clinical lab, such as a CLIA lab?

    Yes, but only after validating the software per institution, local, state, and federal guidelines before using the Illumina BaseSpace Variant Interpreter software.

  • Can I sequence Nextera Rapid Capture Enrichment libraries on the Genome Analyzer IIx?
    Yes. When preparing libraries on the cBot and sequencing on the HiSeq 2000, GAIIx, and in High Output mode on the HiSeq 2500 new primers are required whether performing a non-indexed, single-indexed, or dual-indexed run. Use the TruSeq Dual Index Sequencing Primer Kit for Paired End runs (catalog # PE-121-1003), which is good for a single run and contains the required primers for sequencing (HP10, HP11, HP12). These primers are included with MiSeq and HiSeq 2500 rapid run reagent kits.
  • What are the best index combinations for my TruSeq library prep?

    Refer to the TruSeq Library Prep Pooling Guide for recommendations and guidelines for Illumina sequencing systems that require balanced index combinations.

  • How do I analyze my data using Illumina software?

    If the run is being uploaded to BaseSpace or BaseSpace Onsite, the data can be analyzed using BaseSpace Core App- MethylSeq and BaseSpace Labs App- MethylKit.

    MethylSeq provides alignment analysis and MethylKit provides sample to sample comparison analysis.

    For more information about MethylSeq, see the MethylSeq BaseSpace App Documentation.

    For more information about MethylKit, see the MethylKit BaseSpace Labs page.

  • Is there an on-instrument analysis solution for the MiSeq?

    At the time of kit launch, an off-instrument Local Run Manager is provided for analysis. The off-instrument software is an interim solution until Local Run Manager for MiSeq is released.

  • Can TruSeq Small RNA libraries be used for directional RNA sequencing?

    No, not currently. The Directional mRNA-Seq Sample Preparation protocol would need to be optimized to take the longer adapters into account, as the size difference makes adapter dimers more difficult to distinguish from actual inserts using SPRI or column purifications. Illumina encourages customers to continue to use the Illumina Small RNA v1.5 adapters as specified in the Directional mRNA-Seq Sample Preparation Guide.

  • What are the expected wash volumes following a HiSeq maintenance wash?

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

    • First water wash-SBS positions only, 32 ml; SBS and PE positions, 72 ml
    • NaOH wash-SBS positions only, 16 ml; SBS and PE positions, 36 ml
    • Final water wash-SBS positions only, 32 ml; SBS and PE positions, 72 ml
  • What is the difference between TruSeq Rapid Exome and Nextera Rapid Capture Enrichment?

    TruSeq Rapid Exome is an optimized enrichment workflow that offers tolerance to input DNA variability along with shortened hybridization times. The new and improved TruSeq Rapid Exome kit uses a new and improved tagmentation enzyme.This kit demonstrates higher enrichment and coverage along with improved accuracy of variant calls and oxo Q-score. 

  • How do I allow BaseSpace Variant Interpreter to access my BaseSpace Sequence Hub account?

    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.

  • What can cause my Nextera Rapid Capture Enrichment library to look different than the example shown in the Nextera Rapid Capture Enrichment Guide?

    The quality and quantity of genomic DNA input into the Nextera tagmentation reaction will affect the library size distribution. A larger peak distribution (> 350 bp) can be indicative of > 50 ng genomic DNA input going into the Nextera tagmentation reaction. Conversely, a smaller sample peak distribution (< 225 bp) can be indicative of < 50 ng genomic DNA or fragmented,  low quality genomic DNA.

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

  • How do I merge data from 2 flow cells?

    Using CASAVA: To merge data from different flow cells (different runs), use the configureBuild script in CASAVA v1.8.2. First, align the data (samples) from each flow cell separately using configureAlignment. Then, include each sample directory as an input directory in the configureBuild.pl command line. Input directories are specified by the -id option, as detailed on page 100 of the CASAVA v1.8.2 User Guide (Rev C).

    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.

  • What is a matrix file?
    The matrix file is used for base calling and accounts for cross talk between dyes.
  • Is a sample sheet required for a sequencing run on the NextSeq system?

    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.

  • How does the data quality compare between the NextSeq 550 system and NextSeq 500 system?

    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.

  • Why is fragmentation buffer used instead of nebulization or sonication?
    This fragmentation method takes advantage of the sensitivity of RNA to cleavage by divalent cations and temperature. This is the most robust method for fragmentation of RNA. Fragmentation by this method has been shown to result in more uniform sequencing coverage compared with other methods.
  • What read lengths are recommended for sequencing ChIP libraries?
    The TruSeq ChIP Sample Prep Kit generates paired-end libraries that can be run on either single-read or paired-end flow cells. In many cases a single end (1 x 50 bp) run is sufficient for ChIP-Seq analysis. Paired-end reads may increase the ability to align reads and to resolve reads mapping to repeat sequences. Please consult with the literature as well as 3rd party analysis software to determine what type of run will be sufficient for the needs of the experiment.
  • What indexing options are available on the HiSeq 3000?

    The HiSeq 3000/4000 reagent kits support single and dual indexing options. SBS kits includes sufficient reagent for 25 additional cycles for indexed sequencing. The indexing primer provides primers for both single and dual indexing.

  • If a network outage to the central file server storing our runs occurs, can the HiSeq 3000 cache an entire 2 x 150 run?

    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.

  • Does the HiSeq require an uninterruptible power supply (UPS)?
    Illumina provides a region-specific UPS with the HiSeq instrument.
  • Can anti-virus software be installed on the NeoPrep System?

    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.

  • Which Illumina sequencing instruments are compatible with this library prep?

    These libraries can be run on all currently available Illumina sequencing systems.

  • What is an amplicon and what is the size of an amplicon?

    An amplicon is a fragment of DNA that is produced from an amplification event such as PCR. Amplicon sizes are user-selected when creating a project in DesignStudio.

    Amplicon sizes can be 150, 175, or 250 bp in size.

  • Can I pool multiple libraries for a single reaction?

    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.

  • Why do I have to do so many bead washings?
    They are necessary to remove the rRNA.
  • What is the recommended read length for sequencing TruSeq Targeted RNA Expression libraries?
    Sequence TruSeq Targeted RNA Expression samples 1 x 50 with dual indices. To prepare your sample sheet, use the Illumina Experiment Manager.
  • What is the shelf life of NeoPrep library prep kit reagents?

    Guaranteed shelf life is 3 months from date of shipment.

  • How many different libraries can be sequenced on a NextSeq flow cell?

    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.

  • Where is the control lane feature?
    The option to designate a control lane has been removed in HCS v2.2. The software includes optimizations that improve the handling of low-diversity libraries, which eliminates the need for a control lane for matrix and phasing estimates.
  • Are components from my other Nextera kits compatible with the Nextera Enrichment kit?
    No. The Nextera Exome/Custom Enrichment kits contain unique reagents that are not provided in other Nextera kits, including Index 2 primers with an "E" prefix. Index 2 primers from other Nextera sample prep kits should not be used.
  • What if I have a magnet other than the recommended magnet for this library prep?

    Cleanup procedures have only been optimized and validated using the magnetic stand specified in the appropriate library prep reference guide. Comparable performance is not guaranteed when using other magnets. If you choose to use a different magnet, test how long samples need to sit on the magnet, as times can vary from the protocol. 

  • What is the Polyploidy Clustering (PC) Module?

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

  • How is the TruSeq ChIP Sample Prep kit different from the legacy ChIP-Seq Sample Prep kit?

    The TruSeq ChIP Sample Prep kit generates Paired End (PE), indexed libraries that are compatible with all Illumina sequencing platforms and can be multiplexed. The legacy ChIP-Seq Sample Prep kit generates Single Read (SR) and non-indexed libraries that are compatible with Genome Analyzer, HiSeq and HiScanSQ, but not MiSeq.

  • Are other sequencing flow cells compatible with MiSeq?

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

  • What are the safe stopping points in the TruSeq Exome protocol?

    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.

  • How many samples can be run on one flow cell?
    Flow cells are designed for single-use. All eight lanes must be used at the same time. They can be used for the same sample or for dif­ferent samples. You can run eight samples at a time without multiplexing. With multiplexing, you can increase throughput to up to 12 samples per lane or up to 96 samples per flow cell.
  • What are the recommended amplicon sizes for the Nextera XT kit?
    Smaller amplicon inputs into Nextera XT preps typically yield smaller insert size ranges. To maximize recovery of smaller fragments out of the AMPureXP cleanup we recommend > 300 bp to ensure even coverage across the length of the DNA fragment. An expected drop off in sequencing coverage about 50 bp from each distal end of a fragment may be seen. This is because the tagmentation reaction cannot add an adapter right at the distal end of a fragment. For PCR amplicon sequencing this can be easily averted by simply designing your amplicons to be ~100 bases larger than the desired insert to be sequenced.
  • Can RiboZero be used before sample prep and enrichment in the TruSeq RNA Access Library Prep protocol?
    The use of RiboZero before sample prep and enrichment is not necessary and has not been tested by Illumina. The initial RNA input is very low for total RNA (10 ng) and FFPE RNA (20 ng).
  • Can low numbers of indices/pooled samples be run together? What will happen if I run improper index combinations? How do I choose the most appropriate combination of indices?

    If pooling fewer than the number of indices provided in the kit, it is necessary to consider low-plexity index combinations. Color balance during the index read is needed to ensure proper image registration. If there is no signal in one of the color channels (red or green) of the index read, image registration may fail and no base will be called for that cycle. If no base is called, the index read may not be able to be matched to the sequence specified in the sample sheet, and then samples will not be able to be demultiplexed. 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.

  • Can I perform longer than 2 x 76 bp runs?

    Performing 2 x 76 bp runs is recommended for optimal performance with fusion callers. If the paired reads are overlapping, fusion calling may be less efficient.

  • How do I make sure that my HiSeq is ready to send data to BaseSpace?
    To upload data to BaseSpace from a HiSeq, a minimum upstream connection of 10 Mbit/second per instrument is needed. Network speed can be assessed by using free online tools such as www.speedtest.net.
  • What browsers are supported for MiSeq Reporter?

    MiSeq Reporter can be viewed with the following web browsers: Firefox 13.0.1+, IE 11+, and Safari 5.1.7+.

  • What is the recommended FFPE DNA extraction method?

    Illumina recommends the Qiagen QIAamp DNA FFPE Tissue Kit (eg, catalog # 56404)

  • What quantitation methods does Illumina recommend for starting material?
    Double Stranded DNA specific dyes, such as QuBit or picogreen.
  • How do I choose index combinations for my libraries if I only pool a small number of samples?

    For more information, see the Nextera Low Plex Pooling Guidelines Technote.  

  • What operating system is used on the instrument computer?
    The HiSeq instrument computer employs 64-bit Windows Vista.
  • Can pre-TruSeq libraries be run on TruSeq cluster kits?

    Yes, TruSeq cluster kits are backwards-compatible.