HTseq-counts not counting correctly?

Question

[vm.higareda]

Hello

I am doing a DE analysis of Drosophila, after mapping my fasq file to Drosophila genome with hisat2. I am using htseq-count with the gtf file to get the count table.

In the count table, I have the identifier corresponding to 28S rRNA and 18S rRNA, but it has ZERO counts, that can not be possible.

Why I have zero counts? I know that I have ribosomal sequences in my treatments

Answer 1

[GenoMax]

By default counting programs ignore multi-mapping reads which are bound to be the case with rRNA reads (see FAQ on this page).

If you want to count them then use this option --non-unique=all :

--nonunique=<nonunique mode>

    Mode to handle reads that align to or are assigned to more than one feature in the overlap <mode> of choice (see -m option). <nonunique mode> are none and all (default: none)

[lieven.sterck]

and most common RNAseq protocols will likely do an ribosomal RNA depletion step as well (== to exclude those from the sequencing)