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Laboratory procedures to generate viral metagenomes

Abstract

This collection of laboratory protocols describes the steps to collect viruses from various samples with the specific aim of generating viral metagenome sequence libraries (viromes). Viral metagenomics, the study of uncultured viral nucleic acid sequences from different biomes, relies on several concentration, purification, extraction, sequencing and heuristic bioinformatic methods. No single technique can provide an all-inclusive approach, and therefore the protocols presented here will be discussed in terms of hypothetical projects. However, care must be taken to individualize each step depending on the source and type of viral-particles. This protocol is a description of the processes we have successfully used to: (i) concentrate viral particles from various types of samples, (ii) eliminate contaminating cells and free nucleic acids and (iii) extract, amplify and purify viral nucleic acids. Overall, a sample can be processed to isolate viral nucleic acids suitable for high-throughput sequencing in 1 week.

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Figure 1
Figure 2: Tangential-flow filter setup for viral-particle concentration.
Figure 3: Buoyant density centrifugation setup and methods.
Figure 4: Filtration setup for viral enumeration and verification of sample purity.
Figure 5: Epifluorescent microscopy to determine viral-particle concentration and purity.

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Acknowledgements

The establishment of these protocols was funded in part through an NSF Postdoctoral Fellowship DBI-0511948 to R.V.T., an NSF grant MCB-0701984 to M.B. and a Marine Microbial Initiative Grant from the Gordon and Betty Moore Foundation to F.R.

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Correspondence to Rebecca V Thurber.

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Thurber, R., Haynes, M., Breitbart, M. et al. Laboratory procedures to generate viral metagenomes. Nat Protoc 4, 470–483 (2009). https://doi.org/10.1038/nprot.2009.10

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