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  • 50+% of my HiSEQ reads are 3' primer (custom primer used)

    Hi All:
    We've run 7 lanes of Illumina sequencing with the HiSEQ.
    We're sequencing the middle of a PCR product...
    We're using a custom sequencing primer to avoid the common 5' end of the PCR product.
    In many of our lanes, we 'see' about 50% of the reads being the 3' end Illumina adapter (barcode side)...

    If we were just using the common Illumina adapter primer (hybridizing to the 5' end), I'd say I have a huge Illumina primer-dimer issue...
    BUT I am using a custom primer...

    So how does this happen?
    Did I get my forward, non-5'-phosphate PCR primer filled in when A-tailing (without, ligation of adapters has bad efficiency (Separate issue)), and then Illumina primers ligated to it...
    IS this possible>?

    OR Is there regular Illumina sequencing primer contamination in my runs, and the Illumina primer dimer is being sequenced...?

    Or?

    Thanks in advance.

  • #2
    Did you synthesize your adapters with phosphothioate bonds in between the ends of the last two 3' bases?

    This prevents exonuclease digestion and adapter artifacts.

    Comment


    • #3
      My PCR primers for generating the construct BEFORE illumina adapter ligation are just regular primers.
      I used the Illumina DNA sample prep kit, so the primers were Illumina, and hence, should have had that O-bond.

      Comment


      • #4
        I would redesign my PCR primers and cut down on the concentration. If you still get primer artifacts gel purify before adapter ligation.

        I assume you purified by some method before ligating the adapters? If not there will be PCR primers with your product.

        Also, don't design the primers with an A at the end since the T overhang from the adaptor might bind to it.

        Comment

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