counting after aligning miRNA reads to miRBase mature miRNA instead of reference genome

Question

[inah]

I have human miRNA-seq data. In the past I have aligned these data using Bowtie2 to the reference genome (Homo_sapiens.GRCh38.dna.toplevel.fa), and I have subsequently performed counting using featureCounts with the annotation file hsa.gff3 from miRBase. . Now I have aligned the reads to the mature miRNA from miRBase (mature.fa), but when I look at the resulting bam files, the reads have a flag of 4 (segment unmapped) and there is no position information in mature.fa.

So can I use mature.fa as the reference for alignment and if so how is counting performed?

Thanks, Ina

Answer 1

[Tm]

yes, you can map the reads to mature miRNAs and can count the reads mapping onto it using samtools idxstat. Alternatively use can try using tool like mirdeep2 for complete analysis of miRNAs.

Answer 2

[kanika.151]

Pardon my ignorance but why cannot featureCounts pick up the counts from a bam file which has been aligned to mature.fa (downloaded from miRbase) ? Is there a specific thing that it looks for in the bam file?

[kanika.151]

I got the answer at this link: miRna-Seq, featureCounts problem