Hello all, I have a set of lncRNAs identified from a plant species for which I need to perform differential gene expression through DESeq2 package. So when I run featureCounts of "Rsubread" package of R to generate raw read counts of the lncRNAs, the output counts.txt shows read counts "0" for most of the lncRNAs, which shouldn't be the case as I have inputted only those lncRNAs that have FPKM value of greater than 1 and hence are expressed in the samples. My featureCounts command line was :
files=c("Con-1-sorted.sam","Con-2-sorted.sam","Con-3-sorted.sam","Treat-1-sorted.sam","Treat-2-sorted.sam","Treat-3-sorted.sam")
fc=featureCounts(files,annot.ext="expressed-lnc.gtf",isGTFAnnotationFile=TRUE, GTF.attrType="transcript_id", allowMultiOverlap=TRUE, strandSpecific=1, isPairedEnd=TRUE)
write.table(x=data.frame(fc$annotation[,c("GeneID","Length")],fc$counts,stringsAsFactors=FALSE),file="counts.txt",quote=FALSE,sep="t",row.names=FALSE)
The counts.txt output looks like this:
GeneID Length Con.1.sorted.sam Con.2.sorted.sam Con.3.sorted.sam Treat.1.sorted.sam Treat.2.sorted.sam Treat.3.sorted.sam
TCONS_00010754 676 0 0 0 0 0 0
TCONS_00003631 1769 0 0 0 0 0 0
TCONS_00009207 659 0 0 0 0 0 0
TCONS_00000101 283 15 31 17 12 12 9
TCONS_00005923 1184 0 0 0 0 0 0
TCONS_00007573 1205 0 0 0 0 0 0
TCONS_00009271 761 0 0 0 0 0 0
TCONS_00000248 590 0 0 0 0 0 0
TCONS_00000249 864 0 0 0 0 0 0
TCONS_00003762 680 0 0 0 0 0 0
TCONS_00000286 848 0 0 0 0 0 0
TCONS_00000293 533 0 0 0 0 0 0
Am I missing something? Previously I didn't use allowMultiOverlap=TRUE but saw somewhere that this might give actual counts for the genes and it worked for some people. However it didn't work for me. I am new to this field so I don't have much knowledge regarding bioinformatic tools and commands. Any help would be appreciated. Thank you.
