Viruses are not yet the obvious application for sequencing at single-organism resolution, with their small and varying sizes that might make them difficult to isolate or their tiny individual amounts of starting genetic material. Single-virus genomics is however an emerging field, pioneered by researchers such as Prof. Manuel Martinez-Garcia who shared with us his advice on setting up your lab and planning your experimental design.
Main considerations for setting up a single-virus genomics lab
The good news is that you do not need special equipment in your lab if you can use shared equipment in core facilities. According to Prof. Manuel Martinez-Garcia, the initial financial investment to setup up your own lab would cost around € 50K. The main challenge will be to use an existing, performant flow cytometry facility.
“Every single country has a really good flow cytometry facility. For single-cell or single-virus genomics, you really don’t need to buy another a new, super expensive instrument and hire skilled people to take care of it,” explains Prof. Martinez-Garcia.
“However, I think is very important to go to a flow cytometry facility that is very familiar with small organisms. Many flow cytometry facilities work only with eukaryotes, which are elephant-sized compared to prokaryotes. So, if you can, select a facility that is already used to work with prokaryotes. In flow cytometry analysis, the scale is important. We had people using the same system that we use and claiming they did not see anything, but they were not familiar with the scale of prokaryotes.”
What kind of data does single-virus genomics produces? It is a great experiment in parallel with classic metagenomics studies and they complement each other, explains Prof. Manuel Martinez-Garcia in his interview.
How long does it take to set up and perform single-virus genomics?
While the financial investment is on the lower and, Prof. Martinez-Garcia suggests that you carefully evaluate and weigh out the results you are aiming for regarding the time you must invest and figure out the best trade-off. In his case, starting from scratch, it took him around six months to set up the entire lab and the experimental design.
Once the entire protocol is ready though, performing the assay is relatively fast.
“If you have everything set up, I’d say starting from your sample until having the single-virus genome amplified in a plate, all can easily be done within a week,” adds Prof. Manuel Martinez-Garcia. “Staining the viruses with a fluorochrome and sorting the sample by FACS takes a day, amplifying the genome another, and finally a third day to analyze the whole genome amplification data.”
Which fractions should I look at in my environmental sample?
When you work on environmental samples the timing for fractionation and processing of the organisms in the sample is important, otherwise a dynamic ecosystem might stray from the original conditions over time. When it comes to single viruses though, the objective is not always as straightforward as isolating viruses from the rest.
Prof. Manuel Martinez-Garcia clarifies: “The question that you have to ask yourself continuously is: ‘What do you want to answer?’. You can for example try to identify all the viruses that are present in the sample, but f you are more interested in the viruses that are active, the ones actually infecting your cells, then you need to have a look at the free viruses fraction but also at the cell fraction, and look at cell infected by several of those viruses.“
“Not all viruses are infecting at the same time, only a few are active at a time point in a sample. Single-virus genomics gives you the whole picture of viral diversity in a sample but looking at the cells uncovers clues about which ones are actively infecting.”
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How quickly should I analyze my viral samples?
Since viruses do not pack any cellular machinery, any potential stress induced on the sample whether of mechanical, chemical or enzymatic nature is not likely to impact the genetic material of interest, which is a stark difference from living cells. However, there is still a natural decay which rates differ for each virus and storing conditions.
“You are not in a rush to process a viral sample. But in any case, I think it’s important to preserve the sample in a good way, and we have just published a paper to address this very important question. We can work with frozen and even fixed samples, as long as the concentrations of fixative, such as glutaraldehyde, remains low.”
Finding a suitable preservation method might then be a good investment of time and resources to incorporate in the experimental design.
How can I prevent the contamination of my samples?
Because single-virus genomics currently relies on whole genome amplification, rigorously avoiding sample contamination at all times is a mandatory requirement. This signifies elaborating clean cell-sorting procedures, the use of clean fumehoods, and the prior decontamination of reagents among many other measures.
“Some companies out there are producing very good whole genome amplification kits,” suggests Prof. Manuel Martinez-Garcia. “If you do not have to mix your own cocktail of reagents, you can just purchase such a kit, they are being used in prestigious labs and quite reliable. If you have to make your own, then you have to be extremely careful about contamination at every single step of the preparation.”
It is recommended to make use of numerous negative controls for the evaluation of contamination. For example, a PCR amplification step for the 16S rRNA gene with relevant primers which would reveal the presence of bacterial contaminants or just sequencing a blank from whole genome amplification step.
- Martínez Martínez J, Martinez-Hernandez F, Martinez-Garcia M. Single-virus genomics and beyond. Nat Rev Microbiol. 2020 Oct 6. doi: 10.1038/s41579-020-00444-0.
- Garcia-Heredia I, Bhattacharjee AS, Fornas O, Gomez ML, Martínez JM, Martinez-Garcia M. Benchmarking of single-virus genomics: a new tool for uncovering the virosphere. Environ Microbiol. 2021 Mar;23(3):1584-1593. doi: 10.1111/1462-2920.15375.
- Martinez-Hernandez, F., Fornas, O., Lluesma Gomez, M. et al.Single-virus genomics reveals hidden cosmopolitan and abundant viruses. Nat Commun 8, 15892 (2017). https://doi.org/10.1038/ncomms15892.
- Rinke, C., Lee, J., Nath, N. et al.Obtaining genomes from uncultivated environmental microorganisms using FACS–based single-cell genomics. Nat Protoc 9, 1038–1048 (2014). https://doi.org/10.1038/nprot.2014.067.