I will sequence viral nucleic acids using Nextera XT library prep. I was informed by Illumina that the input must be dsDNA, at least 300-bp. I am wondering how to get it. I have a commercial kit to prepare the first strand using the viral RNA as template. My problem is synthetizing the second strand. I have seen a strategy where you must use a number of enzymes (RNAse H, ligase, polymerase), but I understand that this is more adequate when you work with long eukaryotic mRNA. For constructing the first strand, I will use random primes. Could I synthetize the second strand using random primers and only a polymerase (Klenow fragment)? In this case, how I would break the RNA/cDNA duplex? Is it possible (and necessary), to validate each step (first strand, second strand) with a qubit fluorometer?
Also, should I eliminate the viral DNA before the reverse transcription? Or may I keep it, get the cDNA for the viral RNA, and use the same reaction tube, with DNA and cDNA, to prepare a single Nextera XT library?
I know that there are commercial kits that prepare both strands at once, however I just found very expensive options (such as SuperScript™ Double-Stranded cDNA Synthesis Kit). If anyone knows a less expensive alternative, I would appreciate the advice.
Hi Renato!
I was just wondering if you got some solution to your own question? I have the very same problem now, as you had 2 years ago!
Many thanks!!
Hi Renato Pariz Maluta and Földes Katalin,
Did either one of you find a solution yet?
Thanks :-)
Renato Pariz Maluta Thanks for answering. You'd think it shouldn't be that complicated.
We are using a protocol for viral ds-cDNA synthesis, and it works well. see this article:
Article A diverse virome of leafroll-infected Grapevine Unveil by dsRNA
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