Quantifying overlapping RNA-seq paired-end reads

Question

[biofalconch]

Hi all,

I'm working with some sequencing data, and I have noticed that all of my reads overlap with their mate, so I get a concordant alignment, however they are represented as two lines in the sam alignment. But my question is: is it ok if I use the bam file with the two lines of the alignment to produce a count matrix? I was planning to use featureCounts.

Thanks in advance for your comments!

Answer 1

[Devon Ryan]

Yes, that is completely normal and happens all of the time. featureCounts will handle this fine.

Answer 2

[liruiradiant]

Yes, you can use the -p option to get Fragment counts, if you don't do so, you get read counts, which is twice the amount