I have R1.fastq and R2.fastq from RNA-Seq experiment. I'm currently trying to perform differential expression using Salmon and DESeq2.

Since the RNA-Seq was done using stranded library kit, it was under my assumption that R1.fastq contains forward reads and R2.fastq contains reverse reads.

So, I used the following command to run Salmon:

    salmon quant -i <index> -l ISF -1 <R1.fastq> -2 <R2.fastq> -o <output> --gcBias

This resulted in poor mapping rate (~3%) and confused me at first since I saw no issues with the sequencing data (e.g. no rRNA contamination, etc...).

Instead, when I tried with below command, I was able to reach ~90% mapping rate:

    salmon quant -i <index> -l ISR -1 <R1.fastq> -2 <R2.fastq> -o <output> --gcBias

From the documentation, F/R of option ***-l*** tells Salmon which strand a read is coming from given a .fastq file:

> F: read1 comes from the forward strand and read2 comes from the reverse strand
>
> R: read1 comes from the reverse strand and read2 comes from the forward strand

So does this mean that in my R1.fastq, there are reverse strand reads?

Thanks in advance.

> Since the RNA-Seq was done using stranded library kit, it was under my
> assumption that R1.fastq contains forward reads and R2.fastq contains
> reverse reads.

That assumption was wrong, as you can see with your own data.  In Truseq standed kits, read 1 is in the reverse orientation.