Hello, I am very new to rna-seq. I have got the RNA-seq data for bacteria (Hiseq 150*2). From lab it was told that its a directional library (NEB ultra directional kit). Now i assume the data is from Sense/Forward strand.
I have used featureCount with -s 1 and only 1% of the reads were assigned to feature. Then i tried with -s 2 (reverse strand) and >96% of reads were assigned. Could someone explain strand concept in feature count.
Why is it so??
Because that's the way the library prep works, the second strand is not amplified (in the case of dUTP method).
thanks. If i count the data with unstranded option how does that impact the?
In all the excitement it seems you lost a word of your sentence.
If i count the data with unstranded option how does that impact the differential expression analysis/any down stream analysis?
Exons which are antisense will get counted incorrectly or not at all because the reads cannot unambiguously get attributed to one of both transcripts.