Hello, I am new to bioinformatics and I am using featureCounts to quantify RNA-Seq reads but I don't grasp the behaviour and meaning of the strand specific options. I have bam files obtain from pair-end reads.
In particular, according to the official documentation:
- using the "-p" option: fragments will be counted instead of reads
- using the "-s 0" option: non strand specific counting is perfomed
- using the "-s 1" option: strand specific counting is performed
- using the "-s 2" option: strand specific counting is performed, but the strands are reversed.
My questions are:
- What is the actual behaviour of featureCounts with these options?
- With a bam file obtained from pair-end reads, should I always use the "-p" option? If not, why should I use the other ones?
- With my data, I observed that using the "-p" option I get about 70% of assigned alignments and I get about the same amount with the "-s 0" option, while I get 0% with "-s 1 / 2". Is this reasonable?
I checked: https://chipster.csc.fi/manual/library-type-summary.html and https://littlebitofdata.com/en/2017/08/strandness_in_rnaseq/ but it didn't help.