I am doing small RNA seq analysis and I have been using featureCounts to assign reads. There was high % of unassigned ambiguity and when I looked into it further into these I can see that many of the reads that had been denoted as ambiguous could be annotated either as tRNA and piRNA or snoRNA and piRNA (using the -O flag in featureCounts to allow overlap).
I have looked at a few of the small RNA annotation tools that are available and they usually annotate reads in a certain order (e.g. miRNA - rRNA - tRNA- snoRNA - piRNA). Within the tools documentation, I haven't been able to find out why they annotate reads in a certain order, would anyone be able to explain this or suggest anything online I could use to understand it?
Any help will be greatly appreciated!