Proportion of RNAseq reads mapping to rRNA, tRNA, mRNA, and intergenic or antisense regions of the genome?

Question

[mathavanbioinfo]

Dear All,

I am doing Whole Transcriptome Analysis, I have completed quality check, alignment and read quantification. I want to check the percentage of expression of the rRNA, tRNA mRNA in each samples. I also want to check where the reads are mapped intergenic or anti-sense region

Regards Mathavan M

Answer 1

[rodolfo.peacewalker]

Hi,

There are some ways to perform a quality check depending on the annotation of your mapped reads (i.e. lncRNA, miRNA, rRNA, tRNA, CDS...). For this purpose, you could take a survey on RseqQc package and use the read_distribution.py script. You're going to need the bed file of your reference to calculate the percentage of reads mapped to each annotation. Here you will find the user's manual for this option.

Hope that it could help!

Best regards!

Answer 2

[i.sudbery]

Depends on whether you are talking about ribo-depleted total RNAseq or polyA selected RNA-seq, and what your fragment size is.

We usually see around 50% of reads mapping to coding exons in a ribo-depleted sample, around 66% for polyA. Quite a lot of the stuff that isn't coming from exons will be coming from intronic sequence.

I wouldn't expect much to be coming from tRNA because in the most common library prep protocols, tRNAs would be too small to be efficiently captured.

[mathavanbioinfo]

Thank you very much for the response

The size of the read is 150, how we can measure and plot the expression levels of my sample

[i.sudbery]

Rather than the read size, I was referring to the fragment size - that is, the size to which the RNA was fragmented in library preparation. In paired end samples, this would be the distance between the two read pairs.