I have paired-end stranded libraries.Can someone please let me know what should I choose for these 3 options to run HISAT2 and htseq thereafter, given that my inferexperiment result is1±,1-+,2++,2–
Q1. For HISAT2, it aks to specify strand information (Unstranded/FR/RF) and then
Q2. also asks to choose*‘Select the upstream/downstream mate orientations for a valid paired-end alignment against the forward reference strand*’ with options (–fr/–rf/–ff).
Q3. for htseq it asks to choose setting for ‘stranded’ with options (yes/no/reverse).
I used FR and --fr for HISAT2 and obtained 43% overall mapping. Then I used those BAM files to perfomr htseq and selected YES for the ‘stranded’ option. After doing MultiQC, I see 0% reads were assigned.