Bowtie2

[njh219]

Hi all
I'm trying to get bowtie2 to work, but every time I run the program it fails to populate the .fastq file it creates. However, it is able to fully populate the .sam file it creates. What additional info should I provide in order to make some sense of this.

[dpryan]

It would be helpful if you posted the command that you're using (as well as what you mean by "populating" a fastq file, by which you presumably mean that you're passing --un-conc or similar).

[njh219]

bowtie2 -p 8 -N 1 --reorder --no-unal -x (ref) -U (trimmed_output) -S (sam_output) --un (fastq_output)
The sam_output file is being filled with the correct amount of reads; however, the fastq file does not receive any reads within the file (of which I expect several million).
That is the code I am using.

[mastal]

What command are you using to run Bowtie2, and what output are you getting from bowtie?

Is bowtie succesfully completing the run, or is it crashing/running out of memory at some point after starting to write the sam file but before writing the fastq file?

[njh219]

Bowtie2 succesfully completes the run and also successfully identifies the number of reads contained within the reference sequence.
The final output is as follows.
18198205 reads; of these:
18198205 (100%) were unpaired; of these:
7695256 (42%) aligned 0 times
1804945 (9%) aligned exactly 1 time
8698004 (47%) aligned >1 times
57% overall alignment rate

[dpryan]

The 9% unique alignment metric is rather worrying, though perhaps it makes sense for your experiment. Regarding the non-functional --un option, this is actually a known bug. Try not specifying --no-unal.

[njh219]

Actually the 9% is great for my experiment (the 42% is what I'm interested in). Thank you very much for your help.
Going foward should I downgrade my version to 2.07 then?

[dpryan]

Yeah, version 2.0.6 actually (the person that submitted the bug I linked to made a typo). Glad the 9% number worked for you, it would have ruined my day!

[njh219]

Just swapped out 2.06 for 2.10 and still getting the same error--the fastq file is not being populated.
Any other reason this might be occuring?

[njh219]

Sorry, answered my own question, looks like the break occured in 2.06. I'm gonna give 2.05 a shot.

[dpryan]

Sorry, answered my own question, looks like the break occured in 2.06. I'm gonna give 2.05 a shot.

Ah, that or just allow bowtie2 to write the unaligned reads too and then use samtools to create a file of unaligned reads and then just convert that back to fastq.