TruSeq DNA PCR-Free FAQs

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  • General


  • The following library prep and index adapter components are available to order through Illumina. From Illumina, order one catalog number for the library prep component and one catalog number for the index adapter component depending on the number of samples for your experiment.

    Library Prep Component

    Catalog #

    TruSeq PCR-Free (24 samples)

    20015962

    TruSeq PCR-Free (96 samples)

    20015963

     

     

    Index Adapter Option

     

    IDT for Illumina- TruSeq DNA UD Indexes (24 indexes)

    20020590

    IDT for Illumina- TruSeq DNA UD Indexes (96 indexes)

    20020591

    TruSeq DNA CD Indexes- 96 Indexes

    20015949

    TruSeq DNA Single Index Set A- 12 Indexes

    20015960

    TruSeq DNA Single Index Set B- 12 Indexes

    20015961

    Single index adapters are provided in tubes.  Each i7 indexed adapter is paired in its tube with the same universal adapter that contains no (i5) barcode.

    Combinatorial Dual Indexed Adapters are plated pairs of adapters such that every i5 indexed adapter would be paired with each i7 indexed adapter in a arrayed matrix across the plate.

    Unique Dual Indexed Adapters are plated pairs of adapters such that every i5 indexed adapter and every i7 indexed adapter is only used once on a plate. With unique dual index adapters, identification and filtering of indexed hopped reads allow customers to confidently multiplex their samples.

    Illumina recommends using Unique Dual Indexed Adapters for library prep and sequencing on Illumina sequencers.

    TruSeq DNA Single Index Set A and B are methylated and suitable for bisulfite sequencing applications. 

    Yes, TruSeq DNA PCR-Free gives better coverage for regions that are difficult to sequence. See the TruSeq DNA PCR-Free Datasheet and example datasets for more detailed information.

    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.

    The library prep reference 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.

    No, Sample Purification Beads (SPB) that are used for size-selection and clean-up steps are included. 

    We offer two kits to support the following sample numbers.

    Library Prep Component

    Catalog #

    TruSeq PCR-Free (24 samples)

    20015962

    TruSeq PCR-Free (96 samples)

    20015963

  • Protocol


  • 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 reference guidefor 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 reference guide.

    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.

    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.

    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 library failure.

    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. 

    Refer to the Index Adapters Pooling Guide for recommendations and guidelines for Illumina sequencing systems that require balanced index combinations.

    If pooling fewer than the number of indexes 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 Index Adapters Pooling Guide for recommendations and guidelines for Illumina sequencing systems that require balanced index combinations. Illumina Experiment Manager (IEM) gives warnings when generating sample sheets if the index combinations do not meet this requirement.