Type

Journal Article

Authors

Colin Hill
R. Paul Ross
Angela McCann
Marion Dalmasso
Thomas D S Sutton
James A. Nolan
Siobhan A McDonnell
Karen M. Daly
Stephen R. Stockdale
Amanda Forde
and 3 others

Subjects

Microbiology

Topics
room temperature human microbiota viral load gut microbiota high throughput operator bias 16s rrna nucleic acids

Reproducible protocols for metagenomic analysis of human faecal phageomes. (2017)

Abstract 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. 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. 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.
Collections Ireland -> Teagasc -> PubMed

Full list of authors on original publication

Colin Hill, R. Paul Ross, Angela McCann, Marion Dalmasso, Thomas D S Sutton, James A. Nolan, Siobhan A McDonnell, Karen M. Daly, Stephen R. Stockdale, Amanda Forde and 3 others

Experts in our system

1
Colin Hill
University College Cork
Total Publications: 351
 
2
R Paul Ross
Teagasc
Total Publications: 441
 
3
Angela McCann
University College Cork
 
4
Marion Dalmasso
Teagasc
Total Publications: 13
 
5
James A. Nolan
University College Cork
Total Publications: 3
 
6
K. Daly
Teagasc
Total Publications: 12
 
7
Stephen R Stockdale
University College Cork
Total Publications: 11
 
8
Amanda Forde
University College Cork
Total Publications: 3