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  • rflrob
    Member
    • May 2010
    • 50

    bowtie2 --all not reporting multiple hits

    I'm trying to map 50PE Illumina reads to a couple reference genomes, and then sort out later which ones are unambiguously from one species or another. I'm trying to use bowtie2 to do the mapping, with the following options:

    bowtie2 -p 8 --all --no-mixed --local

    And when it runs, it happily reports that I have plenty of reads that align concordantly many times:

    34669986 reads; of these:
    34669986 (100.00%) were paired; of these:
    4165729 (12.02%) aligned concordantly 0 times
    23793250 (68.63%) aligned concordantly exactly 1 time
    6711007 (19.36%) aligned concordantly >1 times

    However, when I check the actual sam file, I find that there are no reads where the "secondary alignment" flag bit is set. In particular,

    $ awk 'and(256, $2)' accepted_hits_unsorted.sam

    returns nothing. Is the --all flag broken? Am I being boneheaded in an obvious way?

    Thanks for the help!

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