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  • Miseq dual index Read1 (i7 index) problem?

    During miseq run, good quaility of read1 and read3 (i5), read4.
    But read2 i7 index is low quality.
    I already found many posting about read3 (i5 index) problem.
    but I can’t find about i7 index failure.
    I use Truseq HT dual index primer for 5 sample.
    Help me please.
    Last edited by Min Jung Lee; 12-20-2017, 09:36 AM.

  • #2
    It could be that i7 indexes are not diverse enough. You can check Illumina pooling guidelines for duel indexed adapters (look at page 21).

    http://sapac.support.illumina.com/co...5042173-01.pdf

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    • #3
      Thank you for your reply.
      but, my library is enough diversity.

      Comment


      • #4
        I am pointing to index diversity not the library. I wonder which wells from adapter plate you have used.

        Comment


        • #5
          What sort of adapters are these that you are using?
          Illumina instruments that prime i5 using the flow cell oligos (including the MiSeq) will always produce i5 sequence from a library molecule. But to produce an i7 read you need one of the i7 indexing primers to anneal in your amplicon. MiSeq's seem to have a fairly high annealing temp for their sequencing primers...

          --
          Phillip

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