Hi,

I have a couple of questions regarding parameter choices for featureCounts. I have stranded 150 bp paired-end reads.

I have currently chosen the following settings. As far as I understand, this will count fragments rather than reads, will ignore chimeric fragments, and will count multimapping reads fractionally (primary alignments only; I have set my STAR mapping to output all alignments with the best score as primary alignments).

featureCounts
-p -B −−countReadPairs -C -F GTF --verbose -M --fraction --primary
-A chromosome_name_alias.txt
-T 4
-s 2
-g gene_id
-a Homo_sapiens.GRCh38.103.gtf
-o counts.txt
input.bam

I am interested in whether you would recommend to adjust the fracOverlap, fracOverlapFeature, minOverlap and nonOverlap parameters. By default, minOverlap is 1 - is this sufficient to confidently assign a read to a feature?

I don't fully understand the maxMOp parameter. Is anyone able to provide a simple explanation?

Many thanks for the suggestions.

Best wishes,

Lucy

I wouldn't recommend changing the default values of -d and -D parameters. They work well in most scenarios. Using the default value of minOverlap should work fine. That will get as many reads counted as possible with sufficient confidence. For more info about the maxMOp parameter, see the users guide.