HTSEQ and hisat2 settings for stranded library

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.

Many thanks,G

read strandness is determined by protocol used for preparation of library. Ideally, your sequencing service provider should give it to you as part of description for sequencing data. HiSAT2 and htseq-count settings are determined by the data. If you don’t have information on strandness of your reads, either ask your sequencing provider or determine it as described in Strandness section of this tutorial: 1: RNA-Seq reads to counts
Good description of strandness is available in another tutorial: De novo transcriptome reconstruction with RNA-Seq
Kind regards,

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Thank you, Igor.