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.