Entering edit mode
10.4 years ago
mark.rose
▴
50
Hello
Seemingly according to the manual, bowtie2 will attempt to align paired-end reads first as pairs but then as unpaired reads if necessary. If this is so, can someone explain the following. (Note, I expect one unpaired alignment)
Thanks
Mark
[rosema1@demeter bowtie2]$ bowtie2 -x 12274_pmi.fa --local -N 0 -L 25 -p 1 -1 s_2_1_25_PMI.fq -2 s_2_2_25_PMI.fq -S s_2_1and2_25_PMI.fq-vs-12274_pmi.fa.sam -I 0 -X 750 --score-min G,5,0 -i S,1,0
278 reads; of these:
278 (100.00%) were paired; of these:
278 (100.00%) aligned concordantly 0 times
0 (0.00%) aligned concordantly exactly 1 time
0 (0.00%) aligned concordantly >1 times
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278 pairs aligned concordantly 0 times; of these:
0 (0.00%) aligned discordantly 1 time
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278 pairs aligned 0 times concordantly or discordantly; of these:
556 mates make up the pairs; of these:
556 (100.00%) aligned 0 times
0 (0.00%) aligned exactly 1 time
0 (0.00%) aligned >1 times
0.00% overall alignment rate
[rosema1@demeter bowtie2]$ bowtie2 -x 12274_pmi.fa --local -N 0 -L 25 -p 1 -U s_2_1_25_PMI.fq,s_2_2_25_PMI.fq -S s_2_1_25_PMI.fq-and-s_2_2_25_PMI.fq-vs-12274_pmi.fa.sam -I 0 -X 750 --score-min G,5,0 -i S,1,0
556 reads; of these:
556 (100.00%) were unpaired; of these:
555 (99.82%) aligned 0 times
0 (0.00%) aligned exactly 1 time
1 (0.18%) aligned >1 times
0.18% overall alignment rate
One possibility is that this is just due to random seeding. It sometimes happens that if you rearrange the reads in a file that you'll then get slightly different results, due presumably to that.
If due to a random process, would you expect repeated execution of the tool would sometimes find this alignment? I have executed these commands several times but always get the same answer. Note there are only 552 paired-end reads in this test dataset.
Hello mark.rose!
It appears that your post has been cross-posted to another site: SEQanswers.
This is typically not recommended as it runs the risk of annoying people in both communities.
It'd only change if you used the
--non-deterministicargument.Just now I tried the paired end alignment 10 times with and without the --non-deterministic flag. No difference. The expected single end alignment is never found unless I align in single end mode and then it is found every time.
Sounds like a bug then. You might submit a bug report.
Anyone have any other ideas. No response from bug list.
It can take quite a while to hear back after submitting a bug report for bowtie2 (weeks easily). You can always just use s different aligner if this is really important to you.
So it seems from the several other still unanswered posts there. As for another aligner, you will note from my command line I am attempting something a bit unusual. I trying to find hits in (100bp) reads that consist, at a minimum, of 25bp of contiguous identity. The entire rest of the read need not match. Not all aligners appear able to do this. Bowtie2 (and Bowtie1) can do this (as least in single end mode). Blast can do this but I have billions of reads so it would be slow going. Any suggestions of aligners capable of this?
For what it's worth, you could use pretty much any aligner if you're willing to do a bit of post-processing. This could be done conveniently with a simple python script using pysam (or C using HTSlib). I could help you write such a script if you need it.