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Going viral: next-generation sequencing applied to phage populations in the human gut

Key Points

  • Methods for purifying virus-like particles (VLPs) from microbial communities, procedures for amplifying the small quantities of DNA that are recovered from VLPs, advances in next-generation sequencing, and a number of new computational approaches have laid the foundations for a 'new age of phage', in which rapid progress is being made in characterizing the viral diversity and virus–bacterial host dynamics in the microbial communities residing in a broad range of habitats, including those associated with our human bodies.

  • Phage genomes have limited sequence conservation, making comparative genomics difficult. There is no conserved phylogenetic marker. Despite these obstacles, new techniques have been developed to classify phages.

  • The phage community in the human gut is complex but appears to be much more stable than those in other habitats, such as the ocean. Patterns of temporal and functional variation are being defined using metagenomics. Gnotobiotic animal models hold promise for further characterization of the role of phages in shaping the properties of the human gut microbiota, including its responses to various perturbations.

  • New insights about the microbial ecology in humans have been gleaned from comparative metagenomics studies and have rekindled an interest in phage therapy. Therapeutic goals may include enhancing the ability of probiotic consortia to establish themselves, and the addition of novel functions to the gut microbiome. Representative preclinical models are needed for proof-of-principle, proof-of-efficacy, dosing and safety tests.

Abstract

Over the past decade, researchers have begun to characterize viral diversity using metagenomic methods. These studies have shown that viruses, the majority of which infect bacteria, are probably the most genetically diverse components of the biosphere. Here, we briefly review the incipient rise of a phage biology renaissance, which has been catalysed by advances in next-generation sequencing. We explore how work characterizing phage diversity and lifestyles in the human gut is changing our view of ourselves as supra-organisms. Finally, we discuss how a renewed appreciation of phage dynamics may yield new applications for phage therapies designed to manipulate the structure and functions of our gut microbiomes.

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Figure 1: Experimental and computational methods for the characterization of the phage populations present in the human gut microbiota.
Figure 2: Potential consequences of a temperate phage life cycle in the human gut.
Figure 3: Potential strategies for phage therapy.

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Acknowledgements

Work from the authors' laboratories that is described in this Review was supported by the US National Institutes of Health (NIH) (grants DK78669, DK30292 and DK70977 to J.I.G. and grant GM095384 to F.L.R.) and by the Crohn's and Colitis Foundation of America. A.R. is the recipient of an International Fulbright Science and Technology Award. N.P.S. is a member of the Washington University Medical Scientist Training Program (MSTP), which is funded by NIH grant GM007200. Owing to space limitations, the authors were not able to cite many wonderful studies that are relevant to the topics covered.

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ACLAME

crAss

Phage SEED

Virome

Glossary

CRISPRs

(Clustered regularly interspaced short palindromic repeats). Widespread genetic systems in bacteria and archaea, consisting of multiple copies of palindromic repeats flanking short spacers of viral or plasmid origin. CRISPR elements provide acquired resistance to foreign DNA.

Virus-like particles

(VLPs). Particles that can be recovered from microbial communities using physical separation methods such as density gradient ultracentrifugation and/or filtration. Purified VLPs have physical characteristics that resemble those of viruses, although their capacity for infection has to be subsequently defined.

Virotype

A taxonomic classification that is typically based on a selected percentage identity threshold among viral reads, rather than on phylogenetic markers.

Multiple displacement amplification

A method for exponential isothermal amplification of a DNA template using φ29 DNA polymerase and random primers. Exponential amplification is achieved by attachment of the polymerase to newly elongated fragments, coupled with the strong displacement activity of the enzyme on extension.

Deep biosphere

The deepest oceanic regions in which life is supported.

Prophages

Temperate phages in a host-incorporated state.

α-diversity

Diversity, whether defined using taxonomic or functional characteristics, within a particular locale (habitat) at a particular moment in time.

β-diversity

Diversity measured between samples or locales at a particular moment in time or over time.

γ-diversity

A combination of alpha and beta diversity.

Pan-genome

The global gene repertoire of a microbial species; defined by sequencing the genomes of isolates of that species obtained from a single or multiple habitats.

Bacterial phylotypes

Taxonomic classifications that are based on phylogenetic markers, classically the 16S rRNA gene. Isolates can be arbitrarily assigned to a species-level phylotype if they share ≥97% sequence identity among their 16S rRNA genes.

Lysogenic

Pertaining to a temperate phage: a state in which linear (1:1) replication is achieved through integration of the phage genome into the chromosome of the bacterial host (or, more rarely, the phage exists as a plasmid within the host cell). The integrated phage transcribes genes that repress lytic action, and in some cases expresses genes that promote the fitness of the bacterial host.

Kill-the-winner virus–bacterial host dynamic

A model for the population dynamics of phage–bacterium interactions; this model postulates that an increase in a host population (the winner) is followed by an increase in its corresponding phage predator, resulting in an increase in the rate at which the winner is killed.

Coliphages

Phages that infect coliform bacteria, in particular Escherichia coli.

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Reyes, A., Semenkovich, N., Whiteson, K. et al. Going viral: next-generation sequencing applied to phage populations in the human gut. Nat Rev Microbiol 10, 607–617 (2012). https://doi.org/10.1038/nrmicro2853

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