htseq-count error message

[roll]

I keep getting the following error message when i use the htseq-count

Error occured when processing SAM input (line 9786565):
'pair_alignments' needs a sequence of paired-end alignments
[Exception type: ValueError, raised in __init__.py:612]

I sorted my sam files using samtools sort -n

On top of this I also get many warnings saying if i am sure sam file is properly sorted or not?

Do you know what is the error message about?

[dpryan]

Did you merge BAM files from both paired-end and single-end alignments?

[roll]

Did you merge BAM files from both paired-end and single-end alignments?

yes, they are all mapped properly. How can i check if there is any other mistakes in the data?

[dpryan]

Just browsing through the htseq-count code, it looks like this error will happen if the program things you have paired-end reads and you then hand it a single-end read. You might just feed to unmerged BAM files separately into htseq-count, possibly summing the resulting file in R. Whether you should sum the counts or treat them separately will depend a bit on how they were created. If the paired-end and single-end data were from different library preps of the same sample and you have equivalent data for other samples in your comparison, then I would recommend simply adding "library_type" (i.e., single or paired-end) as a factor in your statistical model (i.e., keep the counts separate). There's an example of that in the DESeq(2) vignette with the pasilla dataset.