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. 

  • 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.