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Reproducible protocols for metagenomic analysis of human faecal phageomes
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 h...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5892011/ https://www.ncbi.nlm.nih.gov/pubmed/29631623 http://dx.doi.org/10.1186/s40168-018-0446-z |
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author | Shkoporov, Andrey N. Ryan, Feargal J. Draper, Lorraine A. Forde, Amanda Stockdale, Stephen R. Daly, Karen M. McDonnell, Siobhan A. Nolan, James A. Sutton, Thomas D.S. Dalmasso, Marion McCann, Angela Ross, R. Paul Hill, Colin |
author_facet | Shkoporov, Andrey N. Ryan, Feargal J. Draper, Lorraine A. Forde, Amanda Stockdale, Stephen R. Daly, Karen M. McDonnell, Siobhan A. Nolan, James A. Sutton, Thomas D.S. Dalmasso, Marion McCann, Angela Ross, R. Paul Hill, Colin |
author_sort | Shkoporov, Andrey N. |
collection | PubMed |
description | 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 °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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s40168-018-0446-z) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-5892011 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-58920112018-04-11 Reproducible protocols for metagenomic analysis of human faecal phageomes Shkoporov, Andrey N. Ryan, Feargal J. Draper, Lorraine A. Forde, Amanda Stockdale, Stephen R. Daly, Karen M. McDonnell, Siobhan A. Nolan, James A. Sutton, Thomas D.S. Dalmasso, Marion McCann, Angela Ross, R. Paul Hill, Colin Microbiome Research 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 °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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s40168-018-0446-z) contains supplementary material, which is available to authorized users. BioMed Central 2018-04-10 /pmc/articles/PMC5892011/ /pubmed/29631623 http://dx.doi.org/10.1186/s40168-018-0446-z Text en © The Author(s). 2018 Open AccessThis 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. |
spellingShingle | Research Shkoporov, Andrey N. Ryan, Feargal J. Draper, Lorraine A. Forde, Amanda Stockdale, Stephen R. Daly, Karen M. McDonnell, Siobhan A. Nolan, James A. Sutton, Thomas D.S. Dalmasso, Marion McCann, Angela Ross, R. Paul Hill, Colin Reproducible protocols for metagenomic analysis of human faecal phageomes |
title | Reproducible protocols for metagenomic analysis of human faecal phageomes |
title_full | Reproducible protocols for metagenomic analysis of human faecal phageomes |
title_fullStr | Reproducible protocols for metagenomic analysis of human faecal phageomes |
title_full_unstemmed | Reproducible protocols for metagenomic analysis of human faecal phageomes |
title_short | Reproducible protocols for metagenomic analysis of human faecal phageomes |
title_sort | reproducible protocols for metagenomic analysis of human faecal phageomes |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5892011/ https://www.ncbi.nlm.nih.gov/pubmed/29631623 http://dx.doi.org/10.1186/s40168-018-0446-z |
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