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  • Not 100% mapping in output bam?

    Running samtools flagstat on a bam file resulting from alignment with Hisat2 gives:
    Code:
    76075665 + 0 in total (QC-passed reads + QC-failed reads)
    1565341 + 0 secondary
    0 + 0 supplementary
    0 + 0 duplicates
    71435955 + 0 mapped (93.90% : N/A)
    74510324 + 0 paired in sequencing
    37255162 + 0 read1
    37255162 + 0 read2
    64430312 + 0 properly paired (86.47% : N/A)
    67187398 + 0 with itself and mate mapped
    2683216 + 0 singletons (3.60% : N/A)
    2452092 + 0 with mate mapped to a different chr
    2095660 + 0 with mate mapped to a different chr (mapQ>=5)
    Shouldn't there be 100% mapping? I think because of this I also get the warning described below.

    I also notice this warning at the end of an HTSeq-count log file when counting features in the same alignment file above:
    Code:
    Warning: 284233 reads with missing mate encountered.
    Should I be worried about this warning? Note that the same raw reads aligned with Tophat2 had 100% mapping in the respective bam file.

  • #2
    The flagstat output will only show 100% mapped if the aligner doesn't output unaligned reads. Tophat doesn't output unaligned reads, hisat2 does, so this value is meaningless on tophat output.

    The htseq-count warning is probably due to an incorrect flag being set in tophat's output (tophat always had the problem, so I wouldn't be surprised if hisat2 does as well). As an aside, featureCounts is vastly faster than htseq-count.

    Comment


    • #3
      Thanks Devon, helps a lot. I'll make sure to give featureCounts a go.

      Comment

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