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  • markduplicates strange results?

    i have a small bam for testing and found that before and after deduplication, the number reads does not seem to match.
    I used the most recent picard tool kit.

    before remove duplication, there are 178306 reported duplications
    after remove duplication, there are 0

    but, the total number of reads just dropped 1586 (453052 - 451466).

    can anyone give any insights?

    $ samtools flagstat 5.bam
    453052 + 0 in total (QC-passed reads + QC-failed reads)
    178306 + 0 duplicates
    447531 + 0 mapped (98.78%:-nan%)
    453052 + 0 paired in sequencing
    226526 + 0 read1
    226526 + 0 read2
    437506 + 0 properly paired (96.57%:-nan%)
    443874 + 0 with itself and mate mapped
    3657 + 0 singletons (0.81%:-nan%)
    5006 + 0 with mate mapped to a different chr
    4378 + 0 with mate mapped to a different chr (mapQ>=5)

    $ samtools flagstat 5.dedup.bam
    451466 + 0 in total (QC-passed reads + QC-failed reads)
    0 + 0 duplicates
    445945 + 0 mapped (98.78%:-nan%)
    451466 + 0 paired in sequencing
    225684 + 0 read1
    225782 + 0 read2
    436046 + 0 properly paired (96.58%:-nan%)
    442404 + 0 with itself and mate mapped
    3541 + 0 singletons (0.78%:-nan%)
    5002 + 0 with mate mapped to a different chr
    4374 + 0 with mate mapped to a different chr (mapQ>=5)

  • #2
    The question then becomes exactly how you removed the duplicates.

    Comment


    • #3
      I don't know the answer to your question, but it looks like after duplicate removal there is a different number of read1's than read2's. That's not good! If two reads are PCR duplicates, then obviously their mates will be as well. I don't know if that behavior is intentional or a bug (I don't use Picard), but I would not want to do that to my data.

      Comment


      • #4
        @Brian: Note that there's a change in singletons as well.

        Comment


        • #5
          Oh I missed that part. Does Picard actually remove reads?
          I set the remove reads to be true though.

          This bam is subsampling from a large one at .5% ratio using samtools.
          Could samtools not sampling the paired reads?

          Thanks and I will post my cmd once I have my computer...

          Originally posted by Brian Bushnell View Post
          I don't know the answer to your question, but it looks like after duplicate removal there is a different number of read1's than read2's. That's not good! If two reads are PCR duplicates, then obviously their mates will be as well. I don't know if that behavior is intentional or a bug (I don't use Picard), but I would not want to do that to my data.

          Comment


          • #6
            Originally posted by dpryan View Post
            @Brian: Note that there's a change in singletons as well.
            What is the singleton sequence ? It means the single read ? Can you explain it in detail ? Thank you !

            Comment


            • #7
              In this context, a singleton is a pair in which only one of the reads mapped.

              Comment


              • #8
                I have a similar issue because after removing duplicates with Picard, I ran ValidateSamFile and I got a substantial proportion of MATE_NOT_FOUND reads.

                Before removing duplicates (with ValidateSamFile):
                "No errors found"

                After removing duplicates:
                "ERROR:MATE_NOT_FOUND 1072882"

                Comment

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