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  • PCR duplicates questions

    Hi,

    I'm still confused about PCR duplicates removal and have some questions about it.

    1. What is PCR duplicates? Can I say that all reads mapped to the same genome location are PCR duplicates?

    2. Is PCR duplicates removal necessary for mRNA Seq and Genome DNA Seq?

    Thanks a lot!
    Slny

  • #2
    PCR duplicates are sequences of DNA that arise from the same parent molecule throughout the course of many PCR cycles. Thus, after sequencing one of them, you do not learn any new biological information from sequencing more of them as you are just repetitively obtaining the sequence of the same parent molecule.

    If two reads map to the exact same location and have the same sequence, that is evidence they are PCR duplicates. It's also possible that this will occur by random chance, especially if you obtain high coverage. If you use paired end reads, then it's easier to pick out duplicates as both reads have to start at the same location.

    You should definitely remove PCR duplicates as they do not yield more information. In fact, they will artificially give you more information, possibly misrepresenting the actual sample.

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    • #3
      For mRNA Seq, if we remove the PCR duplicates, which actually occurred by random chance, then we will get wrong read counts. Is removal of PCR duplicates also recommended in mRNA Seq?

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      • #4
        Originally posted by slny View Post
        For mRNA Seq, if we remove the PCR duplicates, which actually occurred by random chance, then we will get wrong read counts. Is removal of PCR duplicates also recommended in mRNA Seq?
        Essentially, you're removing them because you can't disambiguate whether the read came from a unique bead source versus PCR. IMO, I removed PCR dups based on start and stop alone, because PCR has an inherent error rate. As before, you can't tell whether the base-differences (mm) came from independent events, or PCR-error.

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        • #5
          Originally posted by slny View Post
          For mRNA Seq, if we remove the PCR duplicates, which actually occurred by random chance, then we will get wrong read counts. Is removal of PCR duplicates also recommended in mRNA Seq?
          Also, I removed PCR duplicates for all applications- even RNA-Seq. Clearly, if you're trying to estimate transcript abundance, or estimate splicing-efficiency then PCR duplicates will have some sort of effect on your results. Now I'm not saying it'll be terrible, but subtle- yes. This of course is a matter of opinion, and I've seen people put up 'ok' arguments both ways. I'd say you'd have to get down into the finer details of your experiment as well as see how much PCR was done.

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          • #6
            If you have paired-end data for RNA-seq, PCR duplicates should be removed. There is a very low probability to get identically mapping paired-end reads and the bias from leaving PCR duplicates will almost certainly be worse than the removal of a few genuine fragments.

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            • #7
              Does removal of PCR duplicates mean that all the reads are removed or only one read is kept?

              If only one read is kept, then it won't influence the de novo assembling result no matter removing PCR duplicates or not. If all the reads are removed, then bias is created.

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              • #8
                One read is kept.

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                • #9
                  Got it. Thanks a lot for all the helps.

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