Thank you! While waiting for your reply, I actually tried igenomes gtf file, it definitely reduces ambiguity , but the multimapping issue still remains
This is the output of featureCounts now
| Assigned | 23399833 |
|---|---|
| Unassigned_Unmapped | 0 |
| Unassigned_MappingQuality | 0 |
| Unassigned_Chimera | 0 |
| Unassigned_FragmentLength | 0 |
| Unassigned_Duplicate | 0 |
| Unassigned_MultiMapping | 37557436 |
| Unassigned_Secondary | 0 |
| Unassigned_NonSplit | 0 |
| Unassigned_NoFeatures | 6348801 |
| Unassigned_Overlapping_Length | 0 |
| Unassigned_Ambiguity | 259191 |