Hello! :)

I have aligned my reads from SmallRNA sequencing with Botwie2 against genome (human, hg38) and with the options --end-to-end --very-sensitive and my reads were almost aligned (about 90%).

Then I used featuresCounts in order to obtain read summarization with the gff3 from miRBase (I have converted it into GTF with gffread):

featureCounts -T 5 -t exon -g transcript_id -a hsa.gtf -o counts.txt file.sam

This is the summary file in output (1):

Assigned    37200
Unassigned_Unmapped 2398401
Unassigned_MappingQuality   0
Unassigned_Chimera  0
Unassigned_FragmentLength   0
Unassigned_Duplicate    0
Unassigned_MultiMapping 0
Unassigned_Secondary    0
Unassigned_Nonjunction  0
Unassigned_NoFeatures   43583950
Unassigned_Overlapping_Length   0
Unassigned_Ambiguity    38946846

The number of assigned is very low.... I tried to use featuresCounts with the complete gtf from Gencode, and I obtained this result (2):

Assigned    33503995
Unassigned_Unmapped 2398401
Unassigned_MappingQuality   0
Unassigned_Chimera  0
Unassigned_FragmentLength   0
Unassigned_Duplicate    0
Unassigned_MultiMapping 0
Unassigned_Secondary    0
Unassigned_Nonjunction  0
Unassigned_NoFeatures   26792865
Unassigned_Overlapping_Length   0
Unassigned_Ambiguity    22271136

Lastly, I have taken the gtf from Gencode and I have eliminated the rows that contained "miRNA". The result (3):

Assigned    31544916
Unassigned_Unmapped 2398401
Unassigned_MappingQuality   0
Unassigned_Chimera  0
Unassigned_FragmentLength   0
Unassigned_Duplicate    0
Unassigned_MultiMapping 0
Unassigned_Secondary    0
Unassigned_Nonjunction  0
Unassigned_NoFeatures   47393496
Unassigned_Overlapping_Length   0
Unassigned_Ambiguity    3629584

As you can see the amount of "Unassigned_Ambiguity" in the case 2 was higher than case 3, and the amount of assigned in case 3 was higher than case 1.

Could you help me to understand what is happening? Are there specific parameters in featuresCount that I have to use with smallRNA data? I was expecting to obtained an higher amount of "Assigned" for miRNA...

Thank you in advance!