TruSeq DNA LT Sample Prep Kit

TruSeq DNA LT Sample Prep Kit Support

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Catalog IDs: FC-121-2001, FC-121-2002

Download Sample Prep Guide

Questions & Answers

  • What is TruSeq Sample Prep Kit and Cluster Kit compatibility?

    Sample Prep Kit

    TruSeq v2, 2.5, and v3 on cBot

    TruSeq v5 on Custer Station

    v1 and v1.5 on cBot

    v4 on Custer Station

    TruSeq DNA/RNA/Enrichment

    Compatible

    Compatible when the following are used:
    - Indexed read primer (part# 1005726)
    - Multiplexing read 2 primer(part# 1005727)

    TruSeq Small RNA

    Compatible

    Compatible for read 1 only when Small RNA v1.0 primer is used (part# 1001375)

    Epicentre Nextera

    Only compatible if Nextera sequencing primers are used or added to TruSeq HP6-HP8

    Only compatible if Nextera sequencing primers are used or added to Illumina primers

    Epicentre ScriptSeq

    Compatible

    Compatible if index read primer and multiplexing read 2 primer are used

    Epicentre ScriptMiner singleplex

    Compatible

    Compatible with small RNA sequencing primer

    Epicentre ScriptMiner multiplex

    Compatible

    Compatible if index read primer and multiplexing read 2 primer are used

    Older DNA or mRNA

    Compatible

    Compatible

    Small RNA v1.0/v1.5

    Compatible for read 1

    Compatible for read 1 only when Small RNA v1.0 primer is used (part# 1001375)

  • 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).

  • Is there a gel-free TruSeq Sample Prep protocol available?

    • Illumina released a gel-free protocol in June 2011 for DNA (targeted resequencing only).
    • The TruSeq RNA protocol includes a gel-free workflow.

  • 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.

  • Will the TruSeq Sample Prep adapter sequences be made available?

    Adapter sequences are provided in the Illumina Adapter Sequence Letter.

  • 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 TruSeq DNA Sample Preparation Guide or TruSeq RNA Sample Preparation Guide for details.

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

    The same controls are used, but at different concentrations.

  • What if I have a magnet other than the one recommended by Illumina for TruSeq Sample Prep

    Cleanup procedures have only been tested and validated using the magnetic stand specified in the TruSeq DNA Sample Preparation Guide or TruSeq RNA Sample Preparation 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.

  • Are Ampure XP beads supplied with the TruSeq Sample Prep kits?

    Ampure XP beads are user-supplied from Beckman-Coulter Genomics. See the TruSeq DNA Sample Preparation Guide or TruSeq RNA Sample Preparation Guide for ordering information.

  • Does Illumina provide nebulizers in the TruSeq DNA Sample Prep kits?

    Nebulizers are not provided, but can be purchased separately through iCom.

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

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

  • What is the input amount required for TruSeq Sample Prep?

    • DNA - 1 μg
    • RNA - 100 ng
    • Small RNA - 1 μg

  • Is quantitation performed before or after pooling of the libraries during 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 TruSeq DNA Sample Preparation Guide or TruSeq RNA Sample Preparation Guide for details.

  • What is the difference between TruSeq DNA and RNA Sample Prep v1 and v2 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

  • How many samples can be processed per TruSeq Sample Prep 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 - 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 - 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 level of indexing is supported for TruSeq DNA and RNA Sample Prep?

    • TruSeq DNA and RNA Sample Prep v1 kits support 12-plex per lane and 96-plex per flow cell.
    • TruSeq DNA and RNA Sample Prep v2 kits support 24-plex per lane and 192-plex per flow cell.

  • What is the cutover plan for TruSeq DNA and RNA from v1 to v2?

    • The V2 kits are available for order now.
    • The V2 kits first customer ship will take place September 1, 2011.
    • TruSeq DNA and RNA v1 kits will be available as Illumina works through existing inventory.
    • All orders will be converted to v2 kits and this is expected to occur in Q4 2011.

  • 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.

  • Can I expect comparable library preparation performance between the TruSeq DNA v2/LT and HT kits?

    There are variances between the TruSeq DNA v2/LT and HT kits that may result in differences in yield and the amount of adapter dimer for the final library. Be sure to take extra care pipetting the AMPure XP beads accurately in the Clean Up PCR step, to minimize the amount of adapter dimer carried through to the final library. Also, note that the AMPure XP bead ratio at this step is optimized for the longer length of the dual-index adapters.

  • Can TruSeq DNA HT (dual-indexed) libraries be used for downstream enrichment using the TruSeq Exome Enrichment or TruSeq Custom Enrichment kits? Are there any differences if the HT dual-indexed libraries are used compared to the TruSeq DNA v2/LT kits?

    Yes, TruSeq DNA HT libraries can be used for downstream enrichment using either the TruSeq Exome Enrichment or TruSeq Custom Enrichment kits. There are slight differences in the design of the v2/LT and HT adapters; therefore, use of the HT adapters may result in slightly lower percent enrichment compared to the v2/LT adapters.

  • Can I use only one of the indices of a dual-indexed library?

    Sample prep requires both indices be present on the library. However, the second index does not need to be read during sequencing. A Single Indexing workflow is supported on HiSeq, HiScanSQ, GA, and MiSeq instruments. Only Index 1 is allowed to be used in the single indexing workflow. Please see the instrument guides for details on the 8-base Single Indexing workflow.

  • Can low numbers of indices/pooled samples be run together? What will happen if I run improper index combinations?

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

  • Can I use the TruSeq HT adapter plate to generate just a few samples at a time?

    The adapter plate is single-use per well and Illumina recommends that it not undergo more than four freeze/thaw cycles. For this reason, Illumina strongly recommends processing at least 24 samples at a time with the TruSeq HT sample prep kit and adapter plate. Even if 24 samples are processed at a time, these 24 samples can be pooled and sequenced in any Illumina supported configuration including low-plex. If it is desired to prepare only a few samples at a time, Illumina recommends purchasing a TruSeq v2/LT sample prep kit.

  • Can I use the TruSeq HT sample prep kits 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 TruSeq HT libraries be combined with other library types in the same flow cell?

    Illumina does support running libraries prepared by different sample prep kits in different lanes of the same flow cell. If this workflow is performed Dual Index Recipes must be used. The second index sequenced for TruSeq LT or v2 libraries will be monotemplate and may result in cluster registration failure because of this. However, the second index will not be used for demultiplexing of these lanes so it will not affect data quality and read 2 will proceed as normal for these libraries. The SAV Indexing tab will not display index information if indices of different lengths are present in the sample sheet loaded at the start of the run.

  • 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 samples are combined with single index samples on the same flow cell, the single indexed samples will have sequence that can be ignored for the second index read. Two sample sheets must be made: one sample sheet for single index lanes and one sample sheet for dual index lanes. Run CASAVA separately with the appropriate --use -bases-mask command for the index reads to demultiplex appropriately. Demultiplexing the HT lanes requires use bases mask for dual indices and uses all 8 bases of the index reads. Demultiplexing the TruSeq LT/v2 lanes requires use bases mask for Index 1 only and using 6 bases. Please see the CASAVA 1.8.2 User Guide for more information.