I am using
featureCounts to quantify my RNA-seq signal over gene regions. I am using it as follows:
For my single-end reads:
featureCounts -t exon -g gene_id -s 0 -O -T 10 -a <.gtf> -o <.counts> <.bam>
For my paired-end reads:
featureCounts -p -t exon -g gene_id -s 0 -O -T 10 -a <.gtf> -o <.counts> <.bam>
Where I am running into something confusing is with the
-s option. I have run these with all options for
-s, with differing proportions of 'successfully assigned reads'.
-s 0 (unstranded) and
-s 2 both assign about ~25% of the reads, however,
-s 1 assigns only ~3% of reads to gene regions. The fact that both 0 and 2 are producing similar proportions of assigned reads/fragments is interesting, and must say something about the library... but I need help determining exactly what that is.
Thanks in advance. Let me know if any additional information is needed.