HTSeq error with bam file input

Question

[akatrib]

I keep getting error messages when submitting HTSeq jobs, using both -

(a) sorted (by chromosomal coordinates) and

(b) unsorted bam files

I made sure to specify the appropriate order in the HTSeq options: -r pos for both sorted and unsorted. I'm re-running samtools now to sort by name, but I'm not sure that's necessary (since you can specify position in the order option.)

Here is the error message I get when specifiying the unsorted bam file:

Error occured when processing SAM input (record #1794713 in file /xx/xx_Aligned.out.bam

I am not sure why it's referring to a sam input. Am I better off converting to sam? Is there anything else I could be missing

And here is the error message I get when specifying the sorted bam file:

Error occured when processing GFF file (line 1611183 of file /xx/GTF/Homo_sapiens.GRCh37.65.gtf)

I'm sure this is a common problem and that there are many posts about this. I am certainly looking into it but in the meantime was hoping to get any feedback if you're familiar with this. Clearly I'm a newbie to this kind of analysis.

Thank you so much!

[andrew.j.skelton73]

Was that the GTF file that you aligned against? How did you do the alignment?

[akatrib]

Andrew,

Thanks for the quick response. Yes. I used both an existing (previously used for cuffdiff analysis and that worked well) and a newly-downloaded GTF file. For the alignment, I used STAR with unsorted bam output and then used samtools to create the sorted bam files (by position).

[andrew.j.skelton73]

Can you amend your post with the commands you've ran?

[akatrib]

~/software/python-2.7/bin/htseq-count -i gene_id $mapping_result/xx_Aligned.out.sorted.bam \
/xx/GTF/Homo_sapiens.Ensembl.GRCh37.72.gtf -r pos -s yes -f bam -q

Since I sorted by chromosomal coordinates, I thought specifying -r pos would be sufficient. Not sure why I would get the GTF processing error though (and I'm using the same gtf file as before). I will try to re-sort by name and use the -r name option to see if that magically helps resolve this.

[WouterDeCoster]

Did you run the command with -f bam? Because sam is the default type.

[akatrib]

Hi! Yes I did. I added the options I used above.

Answer 1

[sergio.arredondo.alonso]

Try to upgrade the library "pysam", it could be a fast solution.

[akatrib]

Hi Sergio,

I'm running the latest version of pysam (0.9.0).