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Old 02-19-2014, 04:20 AM   #1
Location: Urbana Illinois

Join Date: Jan 2013
Posts: 28
Default Error when using HTSeq count

I used Tophat to produce a bam file from paired end RNAseq data, as well as unpaired reads that were retained after trimming. I then sorted by read name and converted the bam to a sam file. When I run HTSeq with the following arguments:

python -m intersection-strict -s yes 13_accepted_hits.sam Sus_scrofa.gtf

I get an error message:

100000 GFF lines processed.
200000 GFF lines processed.
300000 GFF lines processed.
400000 GFF lines processed.
496967 GFF lines processed.
Error occured when processing SAM input (line 4684 of file 13_acc
'pair_alignments' needs a sequence of paired-end alignments
[Exception type: ValueError, raised in]

It looks to me that there are unpaired reads in the sam file that are causing the problem. Do I have to run separate Tophat alignments with the paired-end and unpaired data, then run the sam files through HTSeq and add the resulting counts together? Is there no way to use HTSeq with a both paired and unpaired reads in the same file? Thanks.
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