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Old 03-11-2013, 07:39 AM   #1
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Default bowtie2 gapped alignment

Hi, I am trying to map some short RNA-Seq reads from mouse against genome (mm10)/transcriptome sequences using bowtie2. There is a particular case which I couldn't resolve. Briefly, bowtie2 is not able to find an alignment for a read of length 33 which in fact can align against the genomic sequence with a single gap roughly in the middle of the read sequence.

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When this gap is corrected by hand, then bowtie2 finds the match. To be more specific the particular sequence is from a U2 ncRNA and a tiny fastq format is attached. The second read in the same file is a faux read created by changing the ID of the first one and filling in the gap by hand.
I have tried bowtie2 with several parameters:
-L 10 -D 20 -R 20 -N 0 -i S,1,0.5
-L 10 -D 20 -R 20 -N 1 -i S,1,0.5
-L 10 -D 20 -R 20 -N 0 -i S,1,0.5 --rdg 1,1
-L 6 --score-min=C,-60,0
-L 6 --score-min=C,-100,0

Only in the last one, I started to get some horribly bad misalignments, but not the aimed alignment with one gap. Can someone explain what I am missing? Thanks for any comments/suggestions.
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bowtie2, gapped alignment

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