Reproducible protocols for metagenomic analysis of human faecal phageomes

Andrey N Shkoporov, Feargal J Ryan, Lorraine A Draper, Amanda Forde, Stephen R Stockdale, Karen M Daly, Siobhan A McDonnell, James A Nolan, Thomas D S Sutton, Marion Dalmasso, Angela McCann, R Paul Ross, Colin Hill

Research output: Contribution to journalArticle

13 Citations (Scopus)

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 °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.

LanguageEnglish
Pages68
Number of pages1
JournalMicrobiome
Volume6
Issue number1
DOIs
Publication statusPublished - 10 Apr 2018
Externally publishedYes

Keywords

  • Bacteriophage
  • Human gut microbiome
  • Metagenomics
  • Phageome
  • Virome

ASJC Scopus subject areas

  • Microbiology
  • Microbiology (medical)

Cite this

Shkoporov, A. N., Ryan, F. J., Draper, L. A., Forde, A., Stockdale, S. R., Daly, K. M., ... Hill, C. (2018). Reproducible protocols for metagenomic analysis of human faecal phageomes. Microbiome, 6(1), 68. https://doi.org/10.1186/s40168-018-0446-z
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. In: Microbiome. 2018 ; Vol. 6, No. 1. pp. 68.
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Shkoporov, AN, Ryan, FJ, Draper, LA, Forde, A, Stockdale, SR, Daly, KM, McDonnell, SA, Nolan, JA, Sutton, TDS, Dalmasso, M, McCann, A, Ross, RP & Hill, C 2018, 'Reproducible protocols for metagenomic analysis of human faecal phageomes', Microbiome, vol. 6, no. 1, pp. 68. https://doi.org/10.1186/s40168-018-0446-z

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.

In: Microbiome, Vol. 6, No. 1, 10.04.2018, p. 68.

Research output: Contribution to journalArticle

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T1 - Reproducible protocols for metagenomic analysis of human faecal phageomes

AU - Shkoporov, Andrey N

AU - Ryan, Feargal J

AU - Draper, Lorraine A

AU - Forde, Amanda

AU - Stockdale, Stephen R

AU - Daly, Karen M

AU - McDonnell, Siobhan A

AU - Nolan, James A

AU - Sutton, Thomas D S

AU - Dalmasso, Marion

AU - McCann, Angela

AU - Ross, R Paul

AU - Hill, Colin

PY - 2018/4/10

Y1 - 2018/4/10

N2 - 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.

AB - 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.

KW - Bacteriophage

KW - Human gut microbiome

KW - Metagenomics

KW - Phageome

KW - Virome

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