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.