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  • hisat2 output more reads than are in the file

    Hi all,
    I am in the middle of testing the hisat2 mapper and encountered a discrepancy between the output hisat2 gives me at the end of the mapping step and the number of reads samtools flagstat counts.

    this is the output I get, when hisat2 is finished:
    Code:
    cat ../hisat2Mapping/WCE7.stat 
    [B]11389273[/B] reads; of these:
      11389273 (100.00%) were paired; of these:
        5961828 (52.35%) aligned concordantly 0 times
        4647893 (40.81%) aligned concordantly exactly 1 time
        779552 (6.84%) aligned concordantly >1 times
        ----
        5961828 pairs aligned concordantly 0 times; of these:
          138165 (2.32%) aligned discordantly 1 time
        ----
        5823663 pairs aligned 0 times concordantly or discordantly; of these:
          11647326 mates make up the pairs; of these:
            10779117 (92.55%) aligned 0 times
            510907 (4.39%) aligned exactly 1 time
            357302 (3.07%) aligned >1 times
    52.68% overall alignment rate
    @Question - how are the 52.68% are calculated? what reads are being considered here as mapped?

    and this is the number of reads I get, when I run samtools flagstat on the sorted/indexed bam file:
    Code:
    samtools flagstat ../hisat2Mapping/WCE7.sorted.bam
    [B]14328481[/B] + 0 in total (QC-passed reads + QC-failed reads)
    2329052 + 0 secondary
    0 + 0 supplementary
    0 + 0 duplicates
    14328481 + 0 mapped (100.00%:-nan%)
    11999429 + 0 paired in sequencing
    6098403 + 0 read1
    5901026 + 0 read2
    10854890 + 0 properly paired (90.46%:-nan%)
    11407498 + 0 with itself and mate mapped
    591931 + 0 singletons (4.93%:-nan%)
    23248 + 0 with mate mapped to a different chr
    18678 + 0 with mate mapped to a different chr (mapQ>=5)
    As you can see, samtools find more reads than there suppose to be originally in the file.

    Is there a simple explanation for that?

    thanks,
    Assa

  • #2
    52.68% = (2*(4647893+779552+138165) + 510907 + 357302)/(2*11389273)

    Samtools is correct, since if sum the numerator you'll get just under 12 million total alignments, which is exactly what's in the BAM file (14328481-2329052).

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