What is the purpose of the second cDNA in strand-specific RNA-seq

Question

[Petesview]

Hi,

This may be a naive question about the dUTP method in strand-specific RNA-seq. I know that mRNA is first being reverse transcribed to make the first strand of cDNA. Subsequently, the first cDNA strand is used as a template to generate the second cDNA strand, but with added dUTP. The two cDNA strands then ligate together but ultimately, the second strand will be digested so that only the first cDNA strand will be amplified.

My question then is, what is the purpose of generating the second cDNA strand with dUTP if it is digested afterwards? Also, in this method, if the first cDNA strand is being amplified via PCR, wouldn't this generate a second cDNA strand, but this time with dNTP hence the lost of strand information? Thank you.

[rfran010]

I think in general for practicality. It's more practical to work with double stranded DNA, and for example with the NEB kit, you do end repair and A-tailing, so that will help with the ligation step and increase its efficiency. I believe ssDNA also requires special ligases or optimized conditions compared to dsDNA.

Answer 1

[Matthias Zepper]

Generally, you should refer to the manual of the kit or the vendor’s support for authoritative answers, because there may be new nifty applications I am unaware of.

But the classic dUTP method was invented in the early 1990ies to prevent cross-sample contamination in PCRs. Because PCR is so sensitive, it basically amplifies even the tiniest trace of suitable contaminant. So if you perform your RNA-seq library preparation in the same room and on the same PCR cycler as your messy colleague before, you might end up with a significant portion of contaminant cDNA in your library. Therefore, larger sequencing facilities usually have separate labs for the pre- and post-PCR steps of a library prep, and it is strictly forbidden to bring anything (samples, pipettes etc.) back.

Using dUTP and uracil-DNA N-glycosylase in the PCR can additionally help to prevent cross-contamination. Two steps differ with regard to basic PCR: dUTP is incorporated into the amplimer instead of dTTP, and the PCR mix is pretreated with uracil-DNA N-glycosylase (UNG). UNG selectively degrades uracil-containing DNA, thus removing any contaminant amplicons from previous PCR reactions. UNG is subsequently heat-inactivated during the PCR and thus the newly synthesized amplicons with dUTP are stable. Only if they happen to be carried over into a new PCR with fresh UNG, they will be degraded.

The second strand is typically created to stabilize the library and prevent the formation of hairpins.