Hello fellow Biostars,
I recently stumbled across the phenomenon that FeatureCounts fails to count fragments instead of reads, despite the command option -p. This only seems to happen when the input BAM file starts with several multialigning reads and contains the tag NH:i:10. The problem then disappears when I remove reads from the first 50-100 lines, if they contain the beforementioned tag.
Do you know why FeatureCounts switches from fragment to the default read count if the beginning of a file has too many reads that align multiple times, e.g. 10x? Does the programme perform some sort of initial checkup?
(I assume that the library preparation had something to do with the abundancy of multialigning reads - I work with RNAseq data from a global RNA library preparation protocol (Illumina Ribo Zero Gold).)
Looking forward to your enlightening answers! :-)