rRNA rate from RNA seq data- Qualimap

Question

[Ahmed.waraky]

Hi

I have a question regarding RNAseq quality control.

I have used Qualimap for RNAseq quality control and wanted to know how can I calculate the rRNA rate (Ratio of all reads aligned to rRNA regions to total uniquely mapped reads) from the alignment reads I got. Or is there any possible way to do that using Qualimap??

Anyone can help?

Thanks!

[opplatek]

Possible duplicate of RNAseq quality control-rRNA rate.

Answer 1

[opplatek]

I don't think comparing rRNA-aligned reads with uniquely mapped reads makes a lot of sense. In general, rRNA mapping reads will be multimapped. One solution to this would be to take only primary mappings, evaluate mapped RNAs, and then calculate ratio of rRNA and other RNAs (protein-coding, for example).

For this, you can use featureCounts, for example (with --primary -M). You can either get gene counts and then assign biotypes or you can go straight to biotypes with -g gene_biotype (assuming your gene annotation has gene_biotype in the attribute field.

Just be careful your gene annotation has all the rRNA annotated correctly. Ensembl, for example, doesn't annotate all rRNAs. I prefer to get rRNA from Silva, map to genome, add to gene annotation, and then estimate rRNA abundance.

[Ahmed.waraky]

Thanks alot, It's required by reviewer, i guess to check for percentage of ribosomal RNA to protein coding-RNA. Do you know if HT-Seq has similar feature or not? I will have to use Silva then as we were using Ensembl.

[opplatek]

I haven't used HTSeq in a long time but according to the manual if you set --type gene_biotype and ----nonunique all --secondary-alignments ignore you should get similar behavior like the one I described for featureCounts. But it would be better if you test it on your own.

[Ahmed.waraky]

Thanks alot :)