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Article Dans Une Revue Scientific Reports Année : 2019

A simple, reproducible and cost- effective procedure to analyse gut phageome: from phage isolation to bioinformatic approach

Résumé

The microbiota of the human gut is a complex and rich community where bacteria and their viruses, the bacteriophages, are dominant. There are few studies on the phage community and no clear standard for isolating them, sequencing and analysing their genomes. Since this makes comparisons between studies difficult, we aimed at defining an easy, low-cost, and reproducible methodology. We analysed five different techniques to isolate phages from human adult faeces and developed an approach to analyse their genomes in order to quantify contamination and classify phage contigs in terms of taxonomy and lifestyle. We chose the polyethylene glycol concentration method to isolate phages because of its simplicity, low cost, reproducibility, and of the high number and diversity of phage sequences that we obtained. We also tested the reproducibility of this method with multiple displacement amplification (MDA) and showed that MDA severely decreases the phage genetic diversity of the samples and the reproducibility of the method. Lastly, we studied the influence of sequencing depth on the analysis of phage diversity and observed the beginning of a plateau for phage contigs at 20,000,000 reads. This work contributes to the development of methods for the isolation of phages in faeces and for their comparative analysis. Our understanding of human gut microbiota has drastically increased in the past decade, but the majority of gut metagenomic studies have focused on the bacterial component of the microbiota in healthy subjects and in patients suffering from various pathological conditions. The human gut virome consists of the whole community of viruses in the gut and is mainly composed of bacteriophages (henceforth called phages) 1. In human gut, there are between 10 9 to 10 12 virus-like particles per gram of faeces 2,3 , a density within the order of magnitude of bacteria. The contribution of phages to gut microbiota ecology and their effects on human host are just beginning to be highlighted 4,5. As an example, an expansion of the number of enteric Caudovirales phages has been observed in patients with Crohn disease and ulcerative colitis 6. The human gut phageome varies according to the age of individuals. The infant phageome is highly dynamic and associated with early life changes in its composition [8]. In contrast, the phageome of adults is stable 7. Healthy adults have a gut phageome mainly constituted of temperate DNA phages belonging to the Caudovirale order (Siphoviridae, Myoviridae, Podoviridae) 3,8. There is a high inter-individual variation between the phageome of healthy individuals 9 , but a recent study showed that a small set of phages are found in the majority of healthy people 10. One of these is the crAssphage, a 97 kbp Podoviridae phage that is highly abundant and ubiquitous in the human gut metagenome 11. Our limited knowledge about gut phages is reflected in the limited host taxonomic
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Dates et versions

hal-02329655 , version 1 (23-10-2019)

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Camille d'Humières, Marie Touchon, Sara Dion, Jean Cury, Amine Ghozlane, et al.. A simple, reproducible and cost- effective procedure to analyse gut phageome: from phage isolation to bioinformatic approach. Scientific Reports, 2019, ⟨10.1038/s41598-019-47656-w⟩. ⟨hal-02329655⟩
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