HISAT2 against miRNA in smallRNA protocol


I am following this protocol (Differential abundance testing of small RNAs) in order to do differential expression of small RNA. There is a section in which reads are aligned against rRNA, and then another section in which reads not aligned to rRNA do HISAT2 against miRNA-hairpin reference sequences. I keep the non-aligned reads. My question is: After this step, is it correct to do HISAT2 against miRNA-mature reference sequences and keep the ones not aligned?
I am following also another protocol for miRNA differential expression. There, they align against mature, hairpin and star reference sequences.

Thank you

Hi @LaiaGutierrez
the answer depends on your goal and definition of small RNA. If you are not interested in miRNAs, use unaligned reads for mapping to small RNAs you interested in. If you are after miRNA expression, use idxstats on BAM (reads mapped to miRNAs) to get counts.
Kind regards,