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Open AccessArticle

A Protocol for Extraction of Infective Viromes Suitable for Metagenomics Sequencing from Low Volume Fecal Samples

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Section of Food Microbiology and Fermentation, Department of Food Science, Faculty of Science, University of Copenhagen, Rolighedsvej 26, 1958 Rederiksberg, Denmark
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Department of Environmental Science, Aarhus University, Frederiksborgvej 399, 4000 Roskilde, Denmark
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Copenhagen Prospective Studies on Asthma in Childhood (COPSAC), Herlev and Gentofte Hospital, University of Copenhagen, Ledreborg Alle 34, 2820 Gentofte, Denmark
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Département de biochimie, de microbiologie, et de bio-informatique, Faculté des sciences et de génie, Université Laval, Québec City, QC G1V 0A6, Canada
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Félix d’Hérelle Reference Center for Bacterial Viruses and Groupe de recherche en écologie buccale, Faculté de médecine dentaire, Université Laval, Québec City, QC G1V 0A6, Canada
*
Authors to whom correspondence should be addressed.
Viruses 2019, 11(7), 667; https://doi.org/10.3390/v11070667
Received: 11 June 2019 / Revised: 10 July 2019 / Accepted: 18 July 2019 / Published: 20 July 2019
(This article belongs to the Special Issue Diversity and Evolution of Phage Genomes)
The human gut microbiome (GM) plays an important role in human health and diseases. However, while substantial progress has been made in understanding the role of bacterial inhabitants of the gut, much less is known regarding the viral component of the GM. Bacteriophages (phages) are viruses attacking specific host bacteria and likely play important roles in shaping the GM. Although metagenomic approaches have led to the discoveries of many new viruses, they remain largely uncultured as their hosts have not been identified, which hampers our understanding of their biological roles. Existing protocols for isolation of viromes generally require relatively high input volumes and are generally more focused on extracting nucleic acids of good quality and purity for down-stream analysis, and less on purifying viruses with infective capacity. In this study, we report the development of an efficient protocol requiring low sample input yielding purified viromes containing phages that are still infective, which also are of sufficient purity for genome sequencing. We validated the method through spiking known phages followed by plaque assays, qPCR, and metagenomic sequencing. The protocol should facilitate the process of culturing novel viruses from the gut as well as large scale studies on gut viromes. View Full-Text
Keywords: human gut phageome; human gut virome; microbiome; isolation; purification; phage; T4; c2; phiX174; phi29 human gut phageome; human gut virome; microbiome; isolation; purification; phage; T4; c2; phiX174; phi29
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MDPI and ACS Style

Deng, L.; Silins, R.; Castro-Mejía, J.L.; Kot, W.; Jessen, L.; Thorsen, J.; Shah, S.; Stokholm, J.; Bisgaard, H.; Moineau, S.; Nielsen, D.S. A Protocol for Extraction of Infective Viromes Suitable for Metagenomics Sequencing from Low Volume Fecal Samples. Viruses 2019, 11, 667.

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