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  • 5' end adapter contamination

    Hey All, I just have a general question. I am dealing with reads generated from illumina and am seeing the 5' adapter in many reads. Why would this occur? would it be a result of mis-annealing of the PCR primer?

    Thanks for any help
    Leanne

  • #2
    Hi Leanne,

    How did you generate your libraries? Which protocol or kit did you use?

    Comment


    • #3
      At least there could be two reasons. If adapter dimers formed during PCR was not cleaned up properly, they will cluster during bridge amplification and sequenced. This type will result in reads which are all adapter sequences followed by predominantly A residues if sequencing cycle was longer than adapter length. Other reason would be where insert length is shorter than cycle number so all or part of adapter is also sequenced.

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      • #4
        Get rid of those reads

        Originally posted by nucacidhunter View Post
        Other reason would be where insert length is shorter than cycle number so all or part of adapter is also sequenced.
        I might be wrong but I think that when this happens it's the 3' that we see with adapter contamination and this is normal. I've also had Illumina sequenced reads come with adapter contamination at the 5' and at the sequencing facility they where unable to explain how this happened. They suggested removing all reads with this contamination. For me the % was low enough to afford losing them against messing up the assembly. I used TRIMMOMATIC to clean up the reads. You can provide an adapter.fasta file and you can include all Illumina adapters just in case. In my reads there was adapter-index-polyA contamination and when I googled the sequence it came up as RNA-Seq adapter and I was doing WGS.

        My libraries were built with Truseq PCR-free kit.

        Regards

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