filtration (large scale, with successive reduction of pore dimension) (or flottation?) followed by evaporation to reduce the volume ?

Herve Levesque Thank you Sir for your response.

But suppose if I have a environment sample which already has low viral particle load. Have you come across any protocol, if yes, please share.

Will be grateful for response. Thank you.

sorry, I don't have any protocol, I make a parallel with archeological research to find cereal remains in sediments which needs to treat large volumes of soil!

No worries thank you !!! Herve Levesque

There are two basic methods: ultracentrifugation at 40 000 G and PEG precipitation. Either way, you will have to filter your sample first, preferably first through a paper filter to remove coarse particles and then through a 0.45 micron filter to remove bacterial cells.

If you have an ultracentrifuge, you can pellet your viruses. 40 000 G for 24 hours should be more than enough.

Most people don't have access to ultracentrifuges these days. In this case, PEG precipitation is the solution. PEG, polyethylene glycol is a high molecular weight linear polymer of hydroxymethyl groups. It will precipitate in conditions of high salt and low temperatures; phages get trapped in the precipitate. Google PEG precipitation of phage and you should find several procedures.

agree with@Herve Levesque

regards

thanks Andrew Jenkins . Will surely do it and let everyone know about my results.

Good luck!

Andrew

filtration with different cut off filters is possible: 0.45 um followed by 0.22um. Ultimately if you want something smaller, you can use 100kDA tangential flow. If most phages you want are dsDNA, then you will be fine with just 0.45 and then 0.22um filters. There is a lot of literature from ocean sampling of that will be useful to read.

Thank you all. As I said I would let you all know about the results, so here it is:

At least for me the results are really surprising. We used four protocols for concentrating Viral/Phage particle, followed by its DNA extraction with viral specific kit and ultimately carrying out NGS to delineate viral diversity of sediment sample.

Note: I have used PES membrane filter of 0.22 micron pore size to get rid of bacteria and microeukaryotes. Also, importantly to get rid of extracellular DNA, I have given DNase treatment and then proceeded with Viral specific DNA extraction kit and without kit method.

The results are surprising because following NGS analysis we are getting only 1 percent reads assigned to VIRUSES and most of the reads assigned to either bacteria or eukaryotes. We also done 16SrRNA gene amplification and as expected only filtrate which was filtered through 0.45 micron got amplified (Although I know, this is a chance event that there should be DNA fragment that should have a stretch of 16SrDNA, but still it does give some hint)

Aren't the result supposed to show only Viral classification, provided that if i have used 0.22 micron filter. This is really confusing.

And also note that my samples, overall, have very less microbial load, so viral load will be even more less.

Note that I have used both PEG precipitation as well Zncl2 pptn to concentrate viral particle.

Please comment.

There is a likelihood that you might have ssDNÀ or RNA viruses that are smaller tajn 0.22 um. Peg is for concentration. In environmental samples, peg has not shown any great results, mostly you can recover dsDNA viruses with the above methods you use. You might see the ssDNA and RNA viruses if they were still intracellular to host cells. Your 10% virus reads should give you a clue to that.

To my knowledge DNAse treatment is suitable to eliminate nuclear DNA not organelle DNA

Hmm ya point taken @Herve Levesque. @Mansa Seth-pasricha, bit confused with your answer, may be I am not able to understand clearly. I have got less than 1 percent reads classified to viruses. And if not PEG, than what should I use(note neither I have ultracentrifugation facility nor I have training with CSCL2 gradient).

Just different filter sizes to concentrate. If you have access to tangential flow filtration, you can filter and also concentrate to a fairly small filter size.

Ok. Thank you Mansha Seth-Pasricha