I used featureCounts to assign features to my mapped reads and I had a varying % ( ~ 20 -65%) of unassigned ambiguity across my files using the command shown below.
featureCounts -T 8 -s 1 *.BAM -a combo_2.gtf -g gene_id -o counts.txt
I then added -O --largestOverlap to the command line to assign reads to overlapping features and across all files there was 100% assigned reads. So my question is for downstream analysis is it ok to use the counts files that includes reads that overlapped with features or is it more appropriate to use the count files where there was no overlapping? (I will be using DESeq2 in downstream analysis)
Also, is there a way to get the reads that weren't assigned in featureCounts into a separate bam file?
Any feedback/help would be great!