Hello,

I'm trying to use featureCounts to annotate a long read dataset obtained from nanopore sequencing. I noticed from the results reads that are supposed to be assigned unambiguously to a specific isoform, were actually assigned all different isoforms, clearly ignoring some isoform-specific splice junctions. One example is shown below, where all the reads were assigned to all 3 ACTG1 isoforms.

I was running featureCounts v2.0.6 in a Conda environment with the following command:

featureCounts -t exon -g transcript_id -O -R CORE -L -a ref.gtf -o counts.tab aln.bam

I was wondering what I can do about it, and I can send the dataset if needed.

Thank you!

The -O option in your command instructs featureCounts to assign a read to all the transcripts that it overlaps by one or more bases. That is why your reads were assigned to all the isoforms. featureCounts does not consider isoform-specific splice junctions when it performs read assignment. It just performs read assignment based on the overlap between reads and targets.