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    Reproducible protocols for metagenomic analysis of human faecal phageomes

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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
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    Keyword
    Human gut microbiome
    Phageome,
    Virome
    Bacteriophages
    Metagenomics
    Date
    2018-04-10
    
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    URI
    http://hdl.handle.net/11019/1542
    Citation
    Shkoporov AN, Ryan FJ, Draper LA, Forde A, Stockdale SR, Daly KM, McDonnell SA, Nolan JA, Sutton TDS, Dalmasso M and others. Reproducible protocols for metagenomic analysis of human faecal phageomes. Microbiome 2018;6(1):68; doi 10.1186/s40168-018-0446-z
    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.
    Funder
    Science Foundation Ireland
    Grant Number
    SFI/12/RC/2273; SFI/14/SP APC/B3032
    ae974a485f413a2113503eed53cd6c53
    https://doi.org/10.1186/s40168-018-0446-z
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    Teagasc publications in Biomed Central

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