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  • Hisat2 reporting of uniquely mapped reads

    Recently i've been optimising Hisat2 to work with the latest version of the human genome. According to its manual, Hisat2 reports the number of valid alignments within the tag NH:i:N, where N is the number of valid alignments.

    At the same time, Hisat2 also outputs an alignment summary at the end of a run, here's an example:

    1000000 reads; of these:
    1000000 (100.00%) were unpaired; of these:
    436566 (43.66%) aligned 0 times
    403452 (40.35%) aligned exactly 1 time
    159982 (16.00%) aligned >1 times
    56.34% overall alignment rate


    I decided to isolate uniquely mapping reads from multimappers and based myself on the NH:i tag, here's the code i used to isolate the uniquely mapping reads:

    cat logs/run0000.o7572309 | awk '{if($2 < 256) print $0}' | grep -P '^@|NH:i:1\b'

    here, the awk section gets rid of all secondary alignments, while grep is supposed to isolate all the reads containing the NH:i:1 tags (i.e. uniquely mapping).

    There seems to be a discrepancy between the number of uniquely mapping reads reported at the end of the alignment:

    403452 (40.35%) aligned exactly 1 time

    and the ones that contain the NH:i:1 tag:

    500082


    is there something i'm overlooking? why the discrepancy?

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