Using HTSeq-count for paired-end data but unsorted by SAMTOOLS

Hi there, as per thread title.

If I am using HTSeq-count on paired-end mapped BAM files, but they are unsorted, and I use -s yes on the default option, is it advisable?

Paired-end .bam need to be sorted either by read name or by alignment position before using HTseq-counts. You can use samtools sort to sort it, then use the -r option in HTseq-counts to specify whether the bam file is sorted by read name (name) or by alignment position (pos).

The -s option of HTseq-counts is completely unrelated to the issue of sorting. It is used to specify whetherf the paired-end data is stranded or not, which depends on sequencing library preparation.

-s <yes/no/reverse>, –stranded=<yes/no/reverse>
whether the data is from a strand-specific assay (default: yes)
For stranded=no, a read is considered overlapping with a feature regardless of whether it is mapped to the same or the opposite
strand as the feature. For stranded=yes and single-end reads, the read
has to be mapped to the same strand as the feature. For paired-end
reads, the first read has to be on the same strand and the second read
on the opposite strand. For stranded=reverse, these rules are
reversed.