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

Abstract

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.

A step-by-step sequence-based analysis of virome enrichment protocol for freshwater and sediment samples

“…The key assumption of the enrichment protocols is that only particles smaller than the minimum filter pore size (0.45 µm and/or 0.22 µm) are able to pass through (22). However, in line with other recent studies (17,18,32,33) Overall, our examination of microbial community diversity and composition associated with the standard virus enrichment protocols, highlight how non-viral particles can be relatively abundant in environmental enriched viromes.…”

“…Filtration is a size-based procedure that is commonly used as a separation step for virome enrichment analysis. Viruses have generally size in diameter between 0.02 µm to 0.4 µm (21,22). The filter's pore size of 0.45 µm and/or 0.22 µm are normally adopted, assuming that only particles smaller than their pore size would pass through the filter and that the resulting filtrate would be therefore free of microbial cells, and enriched with viruses.…”

Fecal Viral Community Responses to High-Fat Diet in Mice

“…S3). However, it important to note that the amplification step (MDA) used in the production of the viromes is known to introduce biases (62,63). The bacterial community changes included increases in Firmicutes with a concomitant decline in Bacteroidetes (Fig.…”

Shining Light on Human Gut Bacteriophages

“…However, this leads to the biased amplification of ssDNA viruses such as Microviridae by as much as 10-fold which in turn skews phage community composition. This is thought to be the confounding factor behind why certain viral datasets have an extremely high relative abundances of Microviridae (Kim et al, 2011 ; Roux et al, 2016 ; McCann et al, 2018 ; d'Humières et al, 2019 ; Garmaeva et al, 2019 ; Shkoporov and Hill, 2019 ; Gregory et al, 2020 ). Many virome studies interpret findings based on relative abundance and thus results may be skewed where MDA has been implemented (Sutton and Hill, 2019 ).…”

“…Many virome studies interpret findings based on relative abundance and thus results may be skewed where MDA has been implemented (Sutton and Hill, 2019 ). MDA has also been shown to affect phage diversity and method reproducibility (d'Humières et al, 2019 ). MDA can introduce GC bias and coverage extremes which can have implications for de novo genome assemblies (Chen et al, 2013 ; Sutton et al, 2019 ).…”