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  • Bowtie2 is unable to align query sequences that contain an intervening stretch of N's

    If I have a reference sequence:
    >reference
    CTAGAATATCACTTAGGCCGGTATTCTATTACTGTACTTCGCGTAAGCTGCTTTAACCC
    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
    GCAAGATTCGCGTGACCCCCACATCCTGGTTCTTCACGAGCAAAGTGTCCTTGCAAATA

    and two query sequences (in a multifasta file):
    >query1
    CTAGAATATCACTTAGGCCGGTATTCTATTACTGTACTTCGCGTAAGCTGCTTTAACCC
    >query2
    GCAAGATTCGCGTGACCCCCACATCCTGGTTCTTCACGAGCAAAGTGTCCTTGCAAATA

    bowtie2 will correctly map the two query sequences onto the reference.

    However, if I change the query to exactly match the reference:
    >query
    CTAGAATATCACTTAGGCCGGTATTCTATTACTGTACTTCGCGTAAGCTGCTTTAACCC
    NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN
    GCAAGATTCGCGTGACCCCCACATCCTGGTTCTTCACGAGCAAAGTGTCCTTGCAAATA

    the query does not map (at all) to the reference.

    The command line parameters are:
    bowtie2 --sensitive-local -x reference.fa -f -U query.fa -S output.sam

    Can anyone explain why this occurs?

  • #2
    Yes, the --n-ceil option defaults to L,0,0.15 which, for your 177 base sequence, is 26 while your read has 59 Ns. You'll probably also have to change -np, which defaults to 1 and could result in the alignment being being below --min-score (I could calculate that, but I'll leave that to you).

    Comment


    • #3
      Ah...thanks so much Devon. Thank god someone has read the manual!...

      Comment

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