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  • TruSeq DNA - need index/barcode-specific primers

    Hi there,
    I have to pool libraries from different sources and labs. One of our collaborator don't have the index information of his library anymore. So I would like to check if sample XY really has the barcode e.g. #12.

    Does someone of you have a primer design suggestion? Or even a Sequence for me?

    Thx,
    Drops

  • #2
    anyone has the same problem?
    Having a indexed library and try to find out which barcode is attached to this library?

    Comment


    • #3
      Log into your Illumina account. There is a document detailing the Illumina primer sequences.

      Comment


      • #4
        Originally posted by drops View Post
        Hi there,
        I have to pool libraries from different sources and labs. One of our collaborator don't have the index information of his library anymore. So I would like to check if sample XY really has the barcode e.g. #12.

        Does someone of you have a primer design suggestion? Or even a Sequence for me?

        Thx,
        Drops
        Personally, if a researcher submitted a library to me and said "Sorry, I forgot what barcode I used." I would be telling that researcher the s/he will be buying for a full lane of sequencing for this library (i.e. the barcode will be irrelevant) because I'm not going to waste my time cleaning up their mess.

        Comment


        • #5
          I agree with kmcarr. The barcode may be identical to one of the other samples, so you cannot safely multiplex the unknown with the others.

          Comment


          • #6
            Sanger Sequence verify the index!

            If I was given a sample that cannot be re-prepped (with known truseq adapters), I would
            1) PCR amplify a small fraction of the sample with small primers at the beginning of the forward universal adapter (say: AATGATACGGCGACCACCGAGATCTAC) and the beginning of the rev adapter (before the truseq index region) (say : GATCGGAAGAGCACACGTCTGAACTCCAG).
            2) Sanger sequence the purified PCR product with the fwd primer. The sample itself will be degenerate (Ns) but you should see the truseq adapter sequence at the end.

            A better strategy is to attach the required adapter by PCR, and run the sequencing experiment!

            Comment

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