Dna Contamination

Question

[Upendra Kumar Devisetty]

I have a basic question about DNA contamination in RNASeq libaries. For reads that are mapped to intron we can always not include them in the analysis but how to deal with those reads that are originated from exons of contaminating DNA?

[Gww]

How do you know that intronic reads are not derived from RNA? or intronic encoded pri-mRNA or lncRNA?

Answer 1

[seidel]

Ever heard of intron retention? For some transcripts splicing of the intron is a regulated event, and introns may be retained until spliced out - thus you may detect intronic reads that are not due to DNA contamination.

The amount of contaminating DNA will depend on how your libraries are constructed. If you use a typical RNA Seq protocol based on an oligo-dT selection step (for simple quantification of gene expression), DNA contamination should be very low. I'm not really sure how you would detect it. I suppose you could try treating a control sample with RNAse to destroy the RNA and then sequence what's left - but I think it would be hard to quantify in a really meaningful way. You could also treat any given RNA prep with DNAse. If you were worried that an analyzed RNA Seq data set had some component that was due to DNA, it would be easier to show that a given signal in that set is resistant to DNAse treatment - and thus you could argue it came from RNA. This would also allow you to see if there are signals that drop out in a DNAse dependent fashion.

One thing to watch out for, is contamination from the genes and plasmids that people work with commonly in the lab. One can often detect high signals in a data set that just happens to come from some gene studied by the post-doc at the next bench over. Common lab molecules are probably easily amplified during some of the library PCR steps.