I can't understand why my featureCounts summary differs so much from the RNA STAR ones. Both were done in Galaxy. I used default parameters (which I can't add here right now because Galaxy has Bad Gateway, but I will add them ASAP). The subsequent PCA plot is not that good, so I suspect that something is wrong with counting.
The featureCounts found 3681407 unassigned unmapped reads, when RNA STAR reported only about 663296 unmapped reads.
What could be the reason?
The example of summary of featureCounts:
Status RNA STAR on data 56, data 38, and data 37: mapped.bam (8,5 weeks, sample 3) Assigned 4705542 Unassigned_Unmapped 3681407 Unassigned_MappingQuality 0 Unassigned_Chimera 0 Unassigned_FragmentLength 0 Unassigned_Duplicate 0 Unassigned_MultiMapping 1557399 Unassigned_Secondary 0 Unassigned_NonSplit 0 Unassigned_NoFeatures 5861961 Unassigned_Overlapping_Length 0 Unassigned_Ambiguity 193083
The RNA STAR result for the same sample:
Number of input reads | 14855335 Average input read length | 478 UNIQUE READS: Uniquely mapped reads number | 10732876 Uniquely mapped reads % | 72.25% Average mapped length | 472.17 Number of splices: Total | 8059241 Number of splices: Annotated (sjdb) | 8025445 Number of splices: GT/AG | 7965722 Number of splices: GC/AG | 72373 Number of splices: AT/AC | 4974 Number of splices: Non-canonical | 16172 Mismatch rate per base, % | 0.44% Deletion rate per base | 0.01% Deletion average length | 1.24 Insertion rate per base | 0.01% Insertion average length | 2.05 MULTI-MAPPING READS: Number of reads mapped to multiple loci | 441052 % of reads mapped to multiple loci | 2.97% Number of reads mapped to too many loci | 67940 % of reads mapped to too many loci | 0.46% UNMAPPED READS: Number of reads unmapped: too many mismatches | 663296 % of reads unmapped: too many mismatches | 4.47% Number of reads unmapped: too short | 2921806 % of reads unmapped: too short | 19.67% Number of reads unmapped: other | 28365 % of reads unmapped: other | 0.19% CHIMERIC READS: Number of chimeric reads | 467140 % of chimeric reads | 3.14%
Any help is highly appreciated!