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  • a problem with RNA-Seq

    Hi All.

    I have problems with RNA-Seq. We used TruSeq kit to prepare libraries and Tophat to align reads. Take the following two genes as examples to show our problems.

    Click image for larger version

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    Almost no reads were mapped to exon 1 for Ptf1a.
    Click image for larger version

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    Many reads were mapped to introns for Rbpjl.

    At beginning, we thought it maybe the problem of alignment software. We tried GSNAP. However, the alignements were similar to Tophat.
    We suspected there might be some random errors in the experiments. However, when checking our another batch of RNA-Seq data which were done two months ago, we found the alignments were replicable between two batches.

    Does anyone have any ideas? Is there anything wrong with our experiments?

    Wish your help! Thanks very much! I appreciate it.

  • #2
    What was the RIN score of your RNA prior to TruSeq RNA prep?

    The lower the RIN score, the more 3' bias you would expect.
    --
    Phillip

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