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

[quokka]

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?

[dpryan]

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).

[quokka]

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