I want to clone the whole viral genome from a water sample from which I have concentrated the virus and done DNA and RNA isolation. I want to find the diversity of viruses and therefore would like to clone the whole genome. How should I go about it?
Dear Vikas
Please find attached paper and a link
http://www.virologyj.com/content/10/1/211 [detailed protocol within this article]
Hope you find this useful
I have never been working with viruses from water. In my short experience with plant virus.
But if you have purified viral particules, in my opinion a "Random PCR" approach will be useful for you, briefly consist in generate a random cDNA library from your encapsidated RNA using NNNNNN plus Universal primer and PCR with a reverse anchored.
I recomend you read this ones:
http://vir.sgmjournals.org/content/88/9/2590.full.pdf
Uhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC336952/pdf/nar00085-0274.pdf
Once you have been generated the library, proceed to sequencing by sanger or maybe NGS technologies (Deep sequencing), in both cases biofinformatic assembly of sequences de novo assembly or mapping against sequences. This same methodology ramdon PCR work fine with dsRNA as an template, example: "dsRNA accumulation is strongly related to plant viruses"
Another methodology I want to suggest to read is siRNA deepsequencing, since few year has become in a very efficent apprach for virus discovery.
Good luck.!
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