Could you please advise me any kits/methods to purify viral DNA from the lobster tissues. Our project aims to sequence whole genomes of several DNA virus species causing disease in lobsters. Size of viral genomes are around 250-500 kb while lobster genome size is around 5 gb.
We only have the infected tissues preserved in alcohol and don't know how to get rid of the host DNA in the gDNA extracts which have DNA of both virus and lobster there. We are looking for a best method to reduce DNA amount of the the host and enrich viral DNA before sequencing and we don't want our NGS data full of the host sequences. There is a kit, NEBNext® Microbiome DNA Enrichment Kit which works well for enrichment of bacteria from vertebrate samples but we don't know would it work for our lobster and virus samples.
Any of your recommendation is highly appreciated. Many thanks.
Hi Joy,
To the best of my knowledge, there is no single kit to purify viral DNA specifically, leaving behind the host DNA. For viral DNA metagenomics, you have to enrich or separate virus particles from the host tissues, essentially before DNA extraction. this includes homogenization, passing through filers of different size, Nuclease treatment, ultracentrifugation and/or PEG precipitation etc. Once you have enriched the virus particles, you have to extract viral DNA from these particles, followed by amplification of the viral DNA using non-specific amplification methods such as RCA, SISPA, VIDISCA , TUViD-VM etc. these techniques help in viral nucleic acid enrichment and host nucleic acid depletion. once you have successfully done this, you can go for NGS studies. Albeit it is very difficult to completely remove the contaminating host DNA, but samples processed through these protocols result in significant increase of the viral sequence reads. Although I am not sure if the viral structures remain intact in alcohol preserved tissues to undergo enrichment protocols. A number of papers are available on virus particle enrichment and non-specific amplification techniques from a number of sources. Please go through them before embarking on your project. I am sharing some literature for your starting reference. None of the papers are on lobster, but you can figure out to process your samples accordingly. One of our reviews can be of some help to you as well (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4534817/).
Best wishes,
SD
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