Questions & Answers

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General

  • 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.

  • 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

  • Are the controls the same in the TruSeq DNA and RNA sample prep kits?

    The same controls are used, but at different concentrations.

  • 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 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.

  • 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.

Input

  • 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.

  • 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

Protocol

  • How do the TruSeq Stranded mRNA and Total RNA protocols generate stranded cDNA?

    Strand specificity is achieved by replacing dTTP with dUTP in the Second Strand Marking Mix (SMM). The incorporation of dUTP in second strand synthesis effectively quenches the second strand during amplification, since the polymerase used in the assay will not incorporate past this nucleotide. Further specificity is achieved by addition of Actinomycin D to the First Strand Master Mix Act D (FSA). Actinomycin prevents spurious DNA dependent synthesis during first strand synthesis, while allowing RNA dependent synthesis.

  • How do I choose index combinations if I only pool a small number of samples with TruSeq sample prep?

    Illumina uses a green laser to sequence G/T bases and a red laser to sequence A/C bases. At each cycle at least one of two nucleotides for each color channel need to be read to ensure proper registration. It is important to maintain color balance for each base of the index read being sequenced, otherwise index read sequencing could fail due to registration failure. For pooling strategies for a small number of samples, please refer to the "Adapter Tube Pooling Guidelines" section in sample prep guide for the kit you are using . Additionally it is recommended to create a sample sheet in the Illumina Experiment Manager (IEM) prior to performing sample prep in order to confirm appropriate index combinations not listed.

  • How 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 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.

  • 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.

  • 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.

  • 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.

  • 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

  • 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.

  • 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.

  • Can I generate larger fragment sizes with TruSeq RNA sample prep protocols?

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

  • Can I 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.

  • 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.

  • 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.

Cluster Generation

  • What cluster generation kit is compatible?

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

  • 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.

Sequencing

  • 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.

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

    The cDNA strand is sequenced.

  • 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).

  • 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.

  • 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.

  • 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

  • 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 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.

Analysis

  • 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.

  • 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 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.

  • 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.