I am using featureCounts to count the number of reads per gene in an RNA-seq experiment. The problem is that I get very low number of assigned reads using featureCounts:
Assigned 3178736 Unassigned_Unmapped 0 Unassigned_MappingQuality 0 Unassigned_Chimera 0 Unassigned_FragmentLength 0 Unassigned_Duplicate 0 Unassigned_MultiMapping 0 Unassigned_Secondary 0 Unassigned_NonSplit 0 Unassigned_NoFeatures 41994410 Unassigned_Overlapping_Length 0 Unassigned_Ambiguity 77571683
I use the following:
featureCounts -M --fraction -g gene_id -T 6 -s 2 -a annotation/hg38.gtf -o counts/mycounts Alignment/*.bam
--The alignment files look fine in IGV. -- Both reference genome and annotation file are from UCSC. -- The library is reversely stranded, as I can also see in IGV.
Thanks in advance for your comments.