I am analysing some public stranded RNA-seq data. Most are paired-end. Some libraries are fr-secondstrand (FR / F) and most are fr-firststrand (RF / R). I have now mapped all libraries with HISAT2 using the correct --rna-strandness parameter (RF, R, F, FR). Now I would like to use featureCount to create a count matrix.
This leads to the question - How should I set these two arguments?
logical indicating if paired-end reads are used. If
TRUE, fragments (templates or read pairs) will be counted instead of individual reads.
FALSE by default.
integer indicating if strand-specific read counting should be performed. It has three possible values: 0(unstranded), 1 (stranded) and 2 (reversely stranded). 0 by default.
My guess is I should create count matrices for the different data types separately and then merge them together. Is this correct?
However, can I simply compare paired end and single end data if I set the parameter isPairedEnd correctly?
And finally strandSpecific=2 means RF / R correct?