dc.contributor.author	Shkoporov, Andrey N.
dc.contributor.author	Ryan, Feargal J.
dc.contributor.author	Draper, Lorraine A.
dc.contributor.author	Forde, Amanda
dc.contributor.author	Stockdale, Stephen R.
dc.contributor.author	Daly, Karen M.
dc.contributor.author	McDonnell, Siobhan A.
dc.contributor.author	Nolan, James A.
dc.contributor.author	Sutton, Thomas D. S.
dc.contributor.author	Dalmasso, Marion
dc.contributor.author	McCann, Angela
dc.contributor.author	Ross, R. Paul
dc.contributor.author	Hill, Colin
dc.contributor.funder	Science Foundation Ireland
dc.contributor.funder	Janssen Biotech
dc.date.accessioned	2018-05-31T11:56:26Z
dc.date.available	2018-05-31T11:56:26Z
dc.description.abstract	Background: Recent studies have demonstrated that the human gut is populated by complex, highly individual and stable communities of viruses, the majority of which are bacteriophages. While disease-specific alterations in the gut phageome have been observed in IBD, AIDS and acute malnutrition, the human gut phageome remains poorly characterised. One important obstacle in metagenomic studies of the human gut phageome is a high level of discrepancy between results obtained by different research groups. This is often due to the use of different protocols for enriching virus-like particles, nucleic acid purification and sequencing. The goal of the present study is to develop a relatively simple, reproducible and cost-efficient protocol for the extraction of viral nucleic acids from human faecal samples, suitable for high-throughput studies. We also analyse the effect of certain potential confounding factors, such as storage conditions, repeated freeze-thaw cycles, and operator bias on the resultant phageome profile. Additionally, spiking of faecal samples with an exogenous phage standard was employed to quantitatively analyse phageomes following metagenomic sequencing. Comparative analysis of phageome profiles to bacteriome profiles was also performed following 16S rRNA amplicon sequencing. Results: Faecal phageome profiles exhibit an overall greater individual specificity when compared to bacteriome profiles. The phageome and bacteriome both exhibited moderate change when stored at + 4 degrees C or room temperature. Phageome profiles were less impacted by multiple freeze-thaw cycles than bacteriome profiles, but there was a greater chance for operator effect in phageome processing. The successful spiking of faecal samples with exogenous bacteriophage demonstrated large variations in the total viral load between individual samples. Conclusions: The faecal phageome sequencing protocol developed in this study provides a valuable additional view of the human gut microbiota that is complementary to 16S amplicon sequencing and/or metagenomic sequencing of total faecal DNA. The protocol was optimised for several confounding factors that are encountered while processing faecal samples, to reduce discrepancies observed within and between research groups studying the human gut phageome. Rapid storage, limited freeze-thaw cycling and spiking of faecal samples with an exogenous phage standard are recommended for optimum results.	en
dc.description.status	Peer reviewed	en
dc.description.version	Published Version	en
dc.format.mimetype	application/pdf	en
dc.identifier.articleid	68
dc.identifier.citation	Shkoporov, A. N., Ryan, F. J., Draper, L. A., Forde, A., Stockdale, S. R., Daly, K. M., McDonnell, S. A., Nolan, J. A., Sutton, T. D. S., Dalmasso, M., McCann, A., Ross, R. P. and Hill, C. (2018) 'Reproducible protocols for metagenomic analysis of human faecal phageomes', Microbiome, 6, 68 (17pp). doi: 10.1186/s40168-018-0446-z	en
dc.identifier.doi	10.1186/s40168-018-0446-z
dc.identifier.endpage	17
dc.identifier.issn	2049-2618
dc.identifier.issued	2018
dc.identifier.journaltitle	Microbiome	en
dc.identifier.startpage	1
dc.identifier.uri	https://hdl.handle.net/10468/6217
dc.identifier.volume	6
dc.language.iso	en	en
dc.publisher	BioMed Central Ltd	en
dc.relation.project	info:eu-repo/grantAgreement/SFI/SFI Research Centres/12/RC/2273/IE/Alimentary Pharmabiotic Centre (APC) - Interfacing Food & Medicine/
dc.relation.project	info:eu-repo/grantAgreement/SFI/SFI Spokes Programme/14/SP APC/B3032/IE/Gut Phageomics - Phage as diagnostics and/or therapeutics in IBD/
dc.relation.uri	https://microbiomejournal.biomedcentral.com/articles/10.1186/s40168-018-0446-z
dc.rights	© 2018, the Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.	en
dc.rights.uri	http://creativecommons.org/licenses/by/4.0/
dc.subject	Human gut microbiome	en
dc.subject	Phageome	en
dc.subject	Virome	en
dc.subject	Bacteriophage	en
dc.subject	Metagenomics	en
dc.title	Reproducible protocols for metagenomic analysis of human faecal phageomes	en
dc.type	Article (peer-reviewed)	en

Reproducible protocols for metagenomic analysis of human faecal phageomes

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