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Laboratory procedures to generate viral metagenomes

Nature Protocols volume 4, pages 470–483 (2009)Cite this article

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.

References

  1. Brussow, H. & Hendrix, R.W. Phage genomics: small is beautiful. Cell 108, 13–16 (2002).

    Article  CAS  Google Scholar 

  2. Angly, F.E. et al. The marine viromes of four oceanic regions. PLoS Biol. 4, 2121–2131 (2006).

    Article  CAS  Google Scholar 

  3. Rohwer, F. & Edwards, R. The phage proteomic tree: a genome-based taxonomy for phage. J. Bacteriol. 184, 4529–4535 (2002).

    Article  CAS  Google Scholar 

  4. Schloss, P.D. & Handelsman, J. Metagenomics for studying unculturable microorganisms: cutting the Gordian knot. Genome. Biol. 6, 229 (2005).

    Article  Google Scholar 

  5. Tringe, S.G. & Rubin, E.M. Metagenomics: DNA sequencing of environmental samples. Nat. Rev. Genet. 6, 805–814 (2005).

    Article  CAS  Google Scholar 

  6. Angly, F. et al. PHACCS, an online tool for estimating the structure and diversity of uncultured viral communities using metagenomic information. BMC Bioinformatics 6, 41 (2005).

    Article  Google Scholar 

  7. Hoffmann, K.H. et al. Power law rank-abundance models for marine phage communities. Fems. Microbiol. Lett. 273, 224–228 (2007).

    Article  CAS  Google Scholar 

  8. Breitbart, M. et al. Diversity and population structure of a near-shore marine-sediment viral community. P. Roy Soc. Lond. B. Bio. 271, 565–574 (2004).

    Article  Google Scholar 

  9. Breitbart, M. et al. Metagenomic analyses of an uncultured viral community from human feces. J. Bacteriol. 185, 6220–6223 (2003).

    Article  CAS  Google Scholar 

  10. Breitbart, M. & Rohwer, F. Method for discovering novel DNA viruses in blood using viral particle selection and shotgun sequencing. Biotechniques 39, 729–736 (2005).

    Article  CAS  Google Scholar 

  11. Breitbart, M. et al. Genomic analysis of uncultured marine viral communities. Proc. Natl. Acad. Sci. USA 99, 14250–14255 (2002).

    Article  CAS  Google Scholar 

  12. Breitbart, M., Wegley, L., Leeds, S., Schoenfeld, T. & Rohwer, F. Phage community dynamics in hot springs. Appl. Environ. Microb. 70, 1633–1640 (2004).

    Article  CAS  Google Scholar 

  13. Desnues, C. et al. Biodiversity and biogeography of phages in modern stromatolites and thrombolites. Nature 452, 340–345 (2008).

    Article  CAS  Google Scholar 

  14. Fierer, N. et al. Metagenomic and small-subunit rRNA analyses reveal the genetic diversity of bacteria, archaea, fungi, and viruses in soil. Appl. Environ. Microb. 73, 7059–7066 (2007).

    Article  CAS  Google Scholar 

  15. Marhaver, K.L., Edwards, R.A. & Rohwer, F. Viral communities associated with healthy and bleaching corals. Environ. Microbiol. 10, 2277–2286 (2008).

    Article  CAS  Google Scholar 

  16. Zhang, T. et al. RNA viral community in human feces: prevalence of plant pathogenic viruses. Plos. Biol. 4, 108–118 (2006).

    Article  CAS  Google Scholar 

  17. Suttle, C.A., Chan, A.M. & Cottrell, M.T. Use of ultrafiltration to isolate viruses from seawater which are pathogens of marine-phytoplankton. Appl. Environ. Microb. 57, 721–726 (1991).

    CAS  Google Scholar 

  18. Steward, G.F., Montiel, J.L. & Azam, F. Genome size distributions indicate variability and similarities among marine viral assemblages from diverse environments. Limnol. Oceanogr. 45, 1697–1706 (2000).

    Article  Google Scholar 

  19. Wommack, K.E., Ravel, J., Hill, R.T., Chun, J.S. & Colwell, R.R. Population dynamics of Chesapeake bay virioplankton: total-community analysis by pulsed-field gel electrophoresis. Appl. Environ. Microb. 65, 231–240 (1999).

    CAS  Google Scholar 

  20. Bench, S.R. et al. Metagenomic characterization of Chesapeake bay virioplankton. Appl. Environ. Microb. 73, 7629–7641 (2007).

    Article  CAS  Google Scholar 

  21. Marie, D., Brussaard, C.P.D., Thyrhaug, R., Bratbak, G. & Vaulot, D. Enumeration of marine viruses in culture and natural samples by flow cytometry. Appl. Environ. Microb. 65, 45–52 (1999).

    CAS  Google Scholar 

  22. Bettarel, Y., Sime-Ngando, T., Amblard, C. & Laveran, H. A comparison of methods for counting viruses in aquatic systems. Appl. Environ. Microb. 66, 2283–2289 (2000).

    Article  CAS  Google Scholar 

  23. Culley, A.I. & Welschmeyer, N.A. The abundance, distribution, and correlation of viruses, phytoplankton, and prokaryotes along a Pacific Ocean transect. Limnol. Oceanogr. 47, 1508–1513 (2002).

    Article  CAS  Google Scholar 

  24. Suttle, C.A. & Feng, C. Mechanisms and rates of decay of marine viruses in seawater. Appl. Environ. Microb. 58, 3721–3729 (1992).

    CAS  Google Scholar 

  25. Noble, R.T. & Fuhrman, J.A. Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria. Aquat. Microb. Ecol. 14, 113–118 (1998).

    Article  Google Scholar 

  26. Wen, K., Ortmann, A.C. & Suttle, C.A. Accurate estimation of viral abundance by epifluorescence microscopy. Appl. Environ. Microb. 70, 3862–3867 (2004).

    Article  CAS  Google Scholar 

  27. Short Protocols in Molecular Biology: a Compendium of Methods from Current Protocols, Vol. 1, 5th edn. (John Wiley and Sons Inc., 2002).

  28. Kunin, V. et al. A bacterial metapopulation adapts locally to phage predation despite global dispersal. Genome Res. 18, 293–297 (2008).

    Article  CAS  Google Scholar 

  29. Schuster, S.C. Next-generation sequencing transforms today's biology. Nat. Methods 5, 16–18 (2008).

    Article  CAS  Google Scholar 

  30. Rohwer, F., Seguritan, V., Choi, D.H., Segall, A.M. & Azam, F. Production of shotgun libraries using random amplification. Biotechniques 31, 108–112 114–116, 118 (2001).

    Article  CAS  Google Scholar 

  31. Wommack, K.E., Bhavsar, J. & Ravel, J. Metagenomics: read length matters. Appl. Environ. Microb. 74, 1453–1463 (2008).

    Article  CAS  Google Scholar 

  32. Wegley, L., Edwards, R., Rodriguez-Brito, B., Liu, H. & Rohwer, F. Metagenomic analysis of the microbial community associated with the coral Porites astreoides . Environ. Microbiol. 9, 2707–2719 (2007).

    Article  CAS  Google Scholar 

  33. Hall, N. Advanced sequencing technologies and their wider impact in microbiology. J. Exp. Biol. 210, 1518–1525 (2007).

    Article  CAS  Google Scholar 

  34. Meyer, M., Stenzel, U. & Hofreiter, M. Parallel tagged sequencing on the 454 platform. Nat. Protoc. 3, 267–278 (2008).

    Article  CAS  Google Scholar 

  35. Allander, T., Emerson, S.U., Engle, R.E., Purcell, R.H. & Bukh, J. A virus discovery method incorporating DNase treatment and its application to the identification of two bovine parvovirus species. Proc. Natl. Acad. Sci. USA 98, 11609–11614 (2001).

    Article  CAS  Google Scholar 

  36. Acheson, N.H. & Tamm, I. Ribonuclease sensitivity of Semliki-Forest virus nucleocapsids. J. Virol. 5, 714–717 (1970).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Chen, K. & Pachter, L. Bioinformatics for whole-genome shotgun sequencing of microbial communities. PLoS Comput. Biol. 1, 106–112 (2005).

    Article  CAS  Google Scholar 

  38. Krause, L. et al. Finding novel genes in bacterial communities isolated from the environment. Bioinformatics 22, E281–E289 (2006).

    Article  CAS  Google Scholar 

  39. Krause, L. et al. Phylogenetic classification of short environmental DNA fragments. Nucleic. Acids Res. 36, 2230–2239 (2008).

    Article  CAS  Google Scholar 

  40. Schloss, P.D. & Handelsman, J. A statistical toolbox for metagenomics: assessing functional diversity in microbial communities. BMC Bioinformatics 9, 34 (2008).

    Article  Google Scholar 

  41. Raes, J., Foerstner, K.U. & Bork, P. Get the most out of your metagenome: computational analysis of environmental sequence data. Curr. Opin. Microbiol. 10, 490–498 (2007).

    Article  CAS  Google Scholar 

  42. Eppley, J.M., Tyson, G.W., Getz, W.M. & Banfield, J.F. Strainer: software for analysis of population variation in community genomic datasets. BMC Bioinformatics 8 (2007).

  43. McHardy, A.C. & Rigoutsos, I. What's in the mix: phylogenetic classification of metagenome sequence samples. Curr. Opin. Microbiol. 10, 499–503 (2007).

    Article  CAS  Google Scholar 

  44. Liolios, K., Mavromatis, K., Tavernarakis, N. & Kyrpides, N.C. The genomes on line database (GOLD) in 2007: status of genomic and metagenomic projects and their associated metadata. Nucleic. Acids Res. 36, D475–D479 (2008).

    Article  CAS  Google Scholar 

  45. Mavromatis, K. et al. Use of simulated data sets to evaluate the fidelity of metagenomic processing methods. Nat. Methods 4, 495–500 (2007).

    Article  CAS  Google Scholar 

  46. Noguchi, H., Park, J. & Takagi, T. MetaGene: prokaryotic gene finding from environmental genome shotgun sequences. Nucleic. Acids Res. 34, 5623–5630 (2006).

    Article  CAS  Google Scholar 

  47. Sommer, D.D., Delcher, A.L., Salzberg, S.L. & Pop, M. Minimus: a fast, lightweight genome assembler. BMC Bioinformatics 8, 64 (2007).

    Article  Google Scholar 

  48. Seshadri, R., Kravitz, S.A., Smarr, L., Gilna, P. & Frazier, M. CAMERA: a community resource for metagenomics. Plos. Biol. 5, 394–397 (2007).

    Article  CAS  Google Scholar 

  49. Markowitz, V.M. Microbial genome data resources. Curr. Opin. Biotechnol. 18, 267–272 (2007).

    Article  CAS  Google Scholar 

  50. Markowitz, V.M. et al. An experimental metagenome data management and analysis system. Bioinformatics 22, E359–E367 (2006).

    Article  CAS  Google Scholar 

  51. Markowitz, V.M. et al. IMG/M: a data management and analysis system for metagenomes. Nucleic Acids Res. 36, D534–D538 (2008).

    Article  CAS  Google Scholar 

  52. Allen, M.J. & Wilson, W.H. Aquatic virus diversity accessed through omic techniques: a route map to function. Curr. Opin. Microbiol. 11, 226–232 (2008).

    Article  CAS  Google Scholar 

  53. Edwards, R.A. & Rohwer, F. Viral metagenomics. Nat. Rev. Microbiol. 3, 504–510 (2005).

    Article  CAS  Google Scholar 

  54. Williamson, K.E., Radosevich, M. & Wommack, K.E. Abundance and diversity of viruses in six Delaware soils. Appl. Environ. Microb. 71, 3119–3125 (2005).

    Article  CAS  Google Scholar 

  55. Thurber, R.V. et al. Metagenomic analysis indicates that stressors induce production of herpes-like viruses in the coral Porites Compressa . P. Natl. Acad. Sci. 105, 18413–18418 (2008).

    Article  Google Scholar 

  56. Arnold, H.P., Ziese, U. & Zillig, W. SNDV, a novel virus of the extremely thermophilic and acidophilic archaeon Sulfolobus. Virology 272, 409–416 (2000).

    Article  CAS  Google Scholar 

  57. Fauquet, C.M., Mayo, M.A., Maniloff, J., Desselberger, U. & Ball, L.A. (eds.) Virus Taxonomy: Classification and Nomenclature of Viruses Vol. VIII. (Elservier, Amsterdam, 2005).

    Google Scholar 

  58. Williamson, K.E., Wommack, K.E. & Radosevich, M. Sampling natural viral communities from soil for culture-independent analyses. Appl. Environ. Microb. 69, 6628–6633 (2003).

    Article  CAS  Google Scholar 

  59. Patel, A. et al. Virus and prokaryote enumeration from planktonic aquatic environments by epifluorescence microscopy with SYBR Green I. Nat. Protoc. 2, 269–276 (2007).

    Article  CAS  Google Scholar 

  60. Koonin, E.V. Virology: Gulliver among the lilliputians. Curr. Biol. 15, R167–R169 (2005).

    Article  CAS  Google Scholar 

  61. Pearson, H. Virophage' suggests viruses are alive. Nature 454, 677–677 (2008).

    Article  CAS  Google Scholar 

  62. Raoult, D. & Forterre, P. Redefining viruses: lessons from Mimivirus. Nat. Rev. Microbiol. 6, 315–319 (2008).

    Article  CAS  Google Scholar 

  63. Van Etten, J.L. & Meints, R.H. Giant viruses infecting algae. Annu. Rev. Microbiol. 53, 447–494 (1999).

    Article  CAS  Google Scholar 

  64. Claverie, J.M. et al. Mimivirus and the emerging concept of 'giant' virus. Virus Res. 117, 133–144 (2006).

    Article  CAS  Google Scholar 

  65. Schoenfeld, T. et al. Assembly of viral metagenomes from Yellowstone hot springs. Appl. Environ. Microb. 74, 4164–4174 (2008).

    Article  CAS  Google Scholar 

  66. Kuwabara, J.S. & Harvey, R.W. Application of a hollow-fiber, tangential-flow device for sampling suspended bacteria and particles from natural-waters. J. Environ. Qual. 19, 625–629 (1990).

    Article  Google Scholar 

  67. Ludwig, K. & Oshaughnessey, K. Tangential-flow filtration—a technical review. Am. Biotechnol. Lab. 7, 41–44 (1989).

    CAS  Google Scholar 

  68. Stang, A., Korn, K., Wildner, O. & Uberla, K. Characterization of virus isolates by particle-associated nucleic acid PCR. J. Clin. Microbiol. 43, 716–720 (2005).

    Article  CAS  Google Scholar 

  69. Sambrook, J., Fritsch, E.F. & Maniatis, T. Molecular Cloning Vol. 1, 2nd edn. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989).

  70. Culley, A.I., Lang, A.S. & Suttle, C.A. The complete genomes of three viruses assembled from shotgun libraries of marine RNA virus communities. Virol. J. 4, 69 (2007).

    Article  Google Scholar 

  71. Culley, A.I., Lang, A.S. & Suttle, C.A. Metagenomic analysis of coastal RNA virus communities. Science 312, 1795–1798 (2006).

    Article  CAS  Google Scholar 

  72. Culley, A.I. & Steward, G.F. New genera of RNA viruses in subtropical seawater, inferred from polymerase gene sequences. Appl. Environ. Microb. 73, 5937–5944 (2007).

    Article  CAS  Google Scholar 

  73. Pinard, R. et al. Assessment of whole genome amplification-induced bias through high-throughput, massively parallel whole genome sequencing. BMC Genomics 7 (2006).

  74. Dean, F.B., Nelson, J.R., Giesler, T.L. & Lasken, R.S. Rapid amplification of plasmid and phage DNA using phi29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res. 11, 1095–1099 (2001).

    Article  CAS  Google Scholar 

  75. Hosono, S. et al. Unbiased whole-genome amplification directly from clinical samples. Genome Res. 13, 954–964 (2003).

    Article  CAS  Google Scholar 

  76. Detter, J.C. et al. Isothermal strand-displacement amplification applications for high-throughput genomics. Genomics 80, 691–698 (2002).

    Article  CAS  Google Scholar 

  77. Ballantyne, K.N., van Oorschot, R.A.H., Muharam, I., van Daal, A. & Mitchell, R.J. Decreasing amplification bias associated with multiple displacement amplification and short tandem repeat genotyping. Anal. Biochem. 368, 222–229 (2007).

    Article  CAS  Google Scholar 

  78. Tomlins, S.A. et al. Whole transcriptome amplification for gene expression profiling and development of molecular archives. Neoplasia 8, 153–162 (2006).

    Article  CAS  Google Scholar 

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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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Authors and Affiliations

  1. Department of Biology, San Diego State University, San Diego, 92182, California, USA

    Rebecca V Thurber, Matthew Haynes, Linda Wegley & Forest Rohwer

  2. Department of Biological Sciences, Florida International University, North Miami, 33138, Florida, USA

    Rebecca V Thurber

  3. College of Marine Science, University of South Florida, Saint Petersburg, 33701, Florida, USA

    Mya Breitbart

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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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