I have noticed a weird discrepancy between the output of featureCounts when run in paired-end mode vs. single end mode on a paired-end sample.
When comparing the percentage of assigned features I get 45% assigned reads for single-end, which seems to be ok from other posts], vs. 5% for paired end mode, the only difference between the two count runs being parameter
-p. I have seen a few posts like this one , reporting low assignment rate, and they mostly seem to be on paired end data, answers seem to suggest that something is wrong with the settings or protocol. However, what if that is not the case?
My data seems ok, with ~100% properly paired reads
Single end counting command:
featureCounts -T64 -s 1 -M -a /export/jonassenfs/michaeld/licebase/genomedata/lsal76ribo/lsalGM.gff3 -o featurecounts-test.txt -g Parent -t exon -B -R 10_S48_R1_001.fastq.gzAligned.sortedByCoord.out.bam > featurecounts.log
Paired-end counting command on the same file yielding only 5% assigned reads:
featureCounts -T64 -p -s 1 -M -a /export/jonassenfs/michaeld/licebase/genomedata/lsal76ribo/lsalGM.gff3 -o featurecounts-test-p.txt -g Parent -t exon -B -R 10_S48_R1_001.fastq.gzAligned.sortedByCoord.out.bam > featurecounts-p.log
Here is the output of samtools flagstat, showing 100% paired reads:
167713978 + 0 in total (QC-passed reads + QC-failed reads) 3459569 + 0 secondary 0 + 0 supplementary 0 + 0 duplicates 167713978 + 0 mapped (100.00% : N/A) 164254409 + 0 paired in sequencing 82127291 + 0 read1 82127118 + 0 read2 164254236 + 0 properly paired (100.00% : N/A) 164254236 + 0 with itself and mate mapped 173 + 0 singletons (0.00% : N/A) 0 + 0 with mate mapped to a different chr 0 + 0 with mate mapped to a different chr (mapQ>=5)