miRNA-seq count by "ID" or "Alias" using mirBase mmu.gff3

Question

[Rafael Soler]

Hello everyone,

I have a conceptual question. Should miRNA reads be counted by the ID term or the Alias term from the mirBase gff3 file?

For example:

15  .   miRNA_primary_transcript    85706603    85706697    .   +   .   ID=MI0000560;Alias=MI0000560;Name=mmu-let-7c-2
15  .   miRNA   85706616    85706637    .   +   .   ID=MIMAT0000523;Alias=MIMAT0000523;Name=mmu-let-7c-5p;Derives_from=MI0000560
15  .   miRNA   85706664    85706685    .   +   .   ID=MIMAT0005439;Alias=MIMAT0005439;Name=mmu-let-7c-2-3p;Derives_from=MI0000560
16  .   miRNA_primary_transcript    77599657    77599750    .   +   .   ID=MI0000559;Alias=MI0000559;Name=mmu-let-7c-1
16  .   miRNA   77599672    77599693    .   +   .   ID=MIMAT0000523_1;Alias=MIMAT0000523;Name=mmu-let-7c-5p;Derives_from=MI0000559
16  .   miRNA   77599717    77599738    .   +   .   ID=MIMAT0004622;Alias=MIMAT0004622;Name=mmu-let-7c-1-3p;Derives_from=MI0000559

Should the HTSeq options be -t=miRNA, -i=Alias or -t=miRNA, -i=ID ? With the first option, the miRNAs that have the same alias are counted, even though they have different genomic positions.

1 - mmu-let-7c-5p 1 - mmu-let-7c-1-3p 1 - mmu-let-7c-2-3p

The second difference between genomic positions. If the objective is to see which miRNAs are affected, what would be the correct option?

1 - mmu-let-7c-5p (genomic position 1) 1 - mmu-let-7c-5p (genomic position 2) 1 - mmu-let-7c-1-3p 1 - mmu-let-7c-2-3p

Thank you!