I'd like to precipitate and enrich for viruses from human samples (BAL, stool, serum, etc.) for metagenomic sequencing. Enrichment is pretty key for some sample types in order to get sufficient virus sequence depth. For example, stool samples will be largely composed of bacteria. Separating the "contaminating" bacterial nucleic acid from the virus nucleic acid is key.
I have a fair amount of experience using protocols similar to published enrichment strategies (the Reyes and Gordon papers, the Breitbart and Rohwer papers, the Bushman papers, etc.) but I'd like something higher throughput.
Any suggestions?
Hi Jason,
In our lab, once we have filtered the samples through 0.45 and 0.2um filters, we do CsCl gradient. Once you have the viral fraction and extracted the DNA, we use genomiphi to have more virus DNA.
http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences/25660030
The protocol is better described in this paper
http://www.nature.com/ismej/journal/vaop/ncurrent/abs/ismej201431a.html
Thanks for your response! I haven't done CsCl, how scalable is it? What volumes of sample do you usually work with? How many samples at a time?
We usually process 1-2mL because they're clinical samples and we don't have access to larger volumes. Our ultracentrifuge can hold up to 4 samples at a time, but it will really depend on the equipment. Some may hold more samples or more volumes. I've also tried precipitation of viruses from environmental samples (up to 1L) using PEG 8000. It usually requires several rounds of precipitation with PEG 8000 until yo can reach a smaller volume. But CsCl should be one the best ways to have highly purified viral particles once you have them concentrated.
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