Nextera DNA Library Prep Kit FAQs

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  • Input


  • Use > 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 might be observed.

    The drop-off occus because the tagmentation reaction cannot add an adapter right at the distal end of a fragment. For PCR amplicon sequencing, averted this issue by designing your amplicons to be ~100 bases larger than the desired insert to be sequenced.

  • Cluster Generation


  • Yes. If you are sequencing combined single-indexed and dual-indexed libraries on a HiSeq, 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. 

  • Sequencing


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

    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.

    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.

    Nextera library 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.

    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.

    • 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 or HiScanSQ 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).

    Combining 12 Index 1 (i7) index adapters and eight Index 2 (i5) index adapters provides 96 dual-index combinations.

    Combining 12 Index 1 (i7) index adapters and eight Index 2 (i5) index adapters provides 96 dual-index combinations.

    Yes. Select the appropriate workflow based on the flow cell type (Single Read or Paired End) when starting your run in HiSeq Control Software (HCS).

  • Analysis


    • HCS v1.5/RTA v1.13/SAV v1.8.4
    • SCS v2.10/RTA v1.13/SAV v1.8.4
    • MiSeq Reporter v1.1
    • CASAVA v1.8.2
    • Illumina Experiment Manager v1.0

    Shorter inserts can lead to sequencing into the adapter. Adapter trimming helps filter out the adapter sequence from the final sequencing data. 

    Select the adapter trimming option when creating a sample sheet in Illumina Experiment Manager (IEM) for use with MiSeq. The MiSeq Reporter (MSR) analysis software automatically trims the adapter sequence. For all other run types, use the adapter trimming option with the appropriate commands.

    For MiSeq runs, a sample sheet is required at the start of the run to enable analysis. For HiSeq 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 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. Illumina recommends creating the sample sheet in the Illumina Experiment Manager (IEM) to confirm appropriate index combinations prior to performing library prep.

    No. To enable demultiplexing, specify the correct use-bases-mask and use the appropriate sample sheet.