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HTseq does not generate any counts file

Here is my code for generating counts from the BAM file.

bsub -J "htseq-count" htseq-count \
-e /scratch/workspace/rnaseq/counts/err.txt \
-o /scratch/workspace/rnaseq/counts/log.out \
-f bam \
-r name \
-s no \
-a 10 \
-t exon \
-i gene_id \
-m intersection-nonempty \
/scratch/workspace/rnaseq/bam/Aligned.sortedByCoord.out.bam \
/scratch/workspace/rnaseq/refs/gencode.v22.annotation.gtf > /scratch/workspace/rnaseq/counts/SRR3088529_counts.txt

But it only generates a txt file which have only one line of information telling the job number and time that I had submitted. Here is the error file.

Traceback (most recent call last):
  File "/scratch/shilpeelab/.local/bin/htseq-count", line 4, in <module>
    __import__('pkg_resources').run_script('HTSeq==0.11.1', 'htseq-count')
  File "/usr/lib/python2.7/site-packages/pkg_resources.py", line 3007, in <module>
    working_set.require(__requires__)
  File "/usr/lib/python2.7/site-packages/pkg_resources.py", line 728, in require
    needed = self.resolve(parse_requirements(requirements))
  File "/usr/lib/python2.7/site-packages/pkg_resources.py", line 626, in resolve
    raise DistributionNotFound(req)
pkg_resources.DistributionNotFound: pysam>=0.9.0

Based on this can you please help me as I am a newbie in this and could not figure out the problem. Thank you.

rna-seq htseq-count
• 2,608 views • link
updated by Ido Tamir Ido Tamir • written by archisman • archisman

You need pysam version 0.9.0 or higher as indicated in the error message. Install it. Instead of show htseq-count you can also use featureCounts which comes as a precompiled binary and is notably faster.

• 0 views • link
written by ATpoint ATpoint

1 answer

You also need pysam on the PYTHONPATH. Normally it should have been installed together with HTSeq-count automatically. Maybe more stuff is broken ... If you work on your own computer you could do this with e.g. pip install pysam https://pypi.org/project/pysam/ . You could also start working with conda https://docs.conda.io/en/latest/ . If its on a shared computing thing you have to ask your adminstrator to fix this for you or you do it with e.g. conda

• 0 views • link
written by Ido Tamir Ido Tamir

Thanks for the reply. I just install pysam today. But it still does not work. I think I have to as my administrator to find the development library for the python.

• 0 views • link
written by archisman • archisman

I guess you use a different python than HTseq count. Best and easiest is you switch to conda and create your own environment independent of the administrators.

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written by Ido Tamir Ido Tamir

Can you please guide me through how to do that?

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written by archisman • archisman

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