RNA contamination in WGS data

Question

[valerie]

Hi guys,

I was wondering if it is possible to see RNAs in paired ended WGS data and didn't manage to find an answer to this question. Does anybody know whether you can see the signal from RNAs in whole genome sequencing data?

Thanks in advance

[WouterDeCoster]

What makes you think there is RNA in your WGS?

[apa@stowers]

Exactly, shouldn't your DNA purification step include RNase treatment?

[valerie]

I am just wondering whether it is possible to detect RNA signal as it is essential for my research not to study RNAs accidentally. There is not only DNA purification step, but also DNA polimerase, that is necessary for DNA replication during sequencing process, that requires DNA or cDNA (for RNAseq) am I right? Is it correct to say that it is simply not possible to sequence RNAs without reverse transcription step that is absent in whole genome sequencing? I am working with Illumina, I suppose that Illumina's DNA polimerase doesn't have reverse transcriptase activity.

[apa@stowers]

That is correct as far as I know.

Answer 1

[igor]

Considering how unstable RNA is, I think it would be difficult to get RNA contamination.

If you just want to spot-check, you can look at the coverage tracks for highly-expressed housekeeping genes. If there is RNA contamination, you should see higher coverage at the exons. WGS coverage should be fairly shallow, so RNA signal would be obvious.

Not sure if there is an easy systematic way to check. Probably the easiest is to compare the average coverage at coding regions to the whole genome.