I am new to RNA-seq and am trying to tabulate featureCounts to feed into DESeq2.
My sequencing is Illumina paired-end directional (dUTP library prep), and I have mapped reads using TopHat (fr firststrand) but when I use featureCounts (Stranded reverse) I am getting the following output:
If I run featureCounts (Unstranded) I get the following:
Isn’t the latter (rather than the former) in line with what I expect? When I view the .bam files in IGV and compare them to the gene annotation files, there are clearly some unannotated genes, but definitely not corresponding to the majority of mapped reads. What have I done wrong? I am using a custom genome, and the gene annotation file I was provided was in .gff3 format, so I have converted it to .gtf using Gffread. Is this the problem? I am dealing with a fungal genome that only rarely has annotated genes overlapping.