How does --primary work with -M and --fractional options featureCounts?

Question

[Lucy]

Hi,

I am trying to understand how the -M, --primary and --fractional work together in featureCounts. From my understanding, -M is ignored if you add the --primary flag. However, what setting should I use if I want to count primary alignments fractionally? Some reads have multiple primary alignments since I have set --outSAMprimaryFlag to AllBestScore in STAR, so all alignments with the best score will be output as primary alignments.

Best wishes,

Lucy

Answer 1

[Istvan Albert]

If you want full control over the alignments then I would recommend filtering the BAM file prior to counting.

In your case make a new BAM file that contains only the primary alignments.

[Lucy]

Yes I could do this and then use -M and --fractional within featureCounts. Is that what you are suggesting?