I was trying to get the counts of miRNAs using htseq-count. The sam file is generated from adapter trimmed miRNA-seq alignment (bowtie) against miRBase mature miRNAs.
The sam file look like this:
HISEQ2000:383:C2452ACXX:1:2316:21334:99296 0 hsa-miR-30c-5p 1 255 23M * 0 0 TGTAAACATCCTACACTCTCAGC @@@DDDD>FFHFBH<C:FB9EE: XA:i:0 MD:Z:23 NM:i:0 HISEQ2000:383:C2452ACXX:1:2316:21266:99322 0 hsa-miR-486-5p 1 255 22M * 0 0 TCCTGTACTGAGCTGCCCCGAG @C@FFFDFFHAHHIIIIIIEED XA:i:0 MD:Z:22 NM:i:0
The reference fasta file used to create bowtie index:
>hsa-let-7a-5p TGAGGTAGTAGGTTGTATAGTT >hsa-let-7a-3p CTATACAATCTACTGTCTTTC
Gtf file used :
hsa-mir-6859-1 . miRNA_primary_transcript 17369 17436 . - . ID "MI0022705"; Alias "MI0022705"; Name "hsa-mir-6859-1" hsa-miR-6859-5p . miRNA 17409 17431 . - . ID "MIMAT0027618"; Alias "MIMAT0027618"; Name "hsa-miR-6859-5p"; Derives_from "MI0022705"
I am not getting any counts for miRNAs based on these files with htseq-count. At the same time, I can see from the sam file that there are mappings to miRNAs.
htseq command used:
htseq-count --mode=intersection-strict --stranded=no --type=miRNA --idattr=Name eg.sam hsa.gff3
Could anyone advise me whether there is anything wrong in this approach or in the files I have used to get the counts ?