Bowtie2 --un-conc output empy

Question

[t2]

Hi all, I am running Bowtie2 to get mapped reads and unmapped reads (--un-conc) from a paired-end experiment.

My command line is like this:

bowtie2 -p 8 --no-unal --un-conc sample1_un.fq -x BDGP6.dna.primary_assembly -U sample1_R1.fastq.gz,sample1_R2.fastq.gz -S sample1.sam

I get a sam file output with content, but the sample1_un.1.fq and sample1_un.2.fq files generated from --un-conc are empty. The output to the screen says 0.67% overall alignment rate so I expect big files in the unmapped reads. The input DNA is human with spike in Drosophila DNA so the alignment rate makes sense.

If anyone could explain what I'm doing wrong that would be great.

Thanks in advance,

T

[Devon Ryan]

Try it without --no-unal and see if that fixes it.

Answer 1

[Asaf]

You give it the input as unpaired (-U), you should give it paired reads with -1 and -2

[t2]

Thanks, Asaf this fixed it. Now I have the files I expect.

I still get empty .1.fq and .2.fq files when I input unpaired F reads (same sample, different lane) using the -U command with and without --no-unal. I still can't figure that part out but for paired end I'm good to go. Thanks for the help!

T

[Asaf]

They unpaired unmapped reads can be caught with --un

[t2]

Of course! the concordant bit doesn't matter with unpaired reads. And Devon Ryan was right, I have to turn the --no-unal switch off in order to get any output.

Thanks!