ena-STUDY-PASTEUR-15-01-2019-13:57:36:821-436
We analysed five different techniques to isolate phages from human adult faeces
The microbiota of the human gut is a complex and rich community where bacteria and their viruses, the phages, are numerically dominant. While the bacterial components have been extensively studied, there are fewer studies on the phage community and no clear standard for isolating the phages and sequencing and analysing their genomes. Since this makes comparisons between studies difficult, we aimed at defining an easy, low-cost, and reproducible methodology for each of these three steps. We analysed five different techniques to isolate phages from human adult faeces and developed a pipeline to analyse their genomes in order to quantify contamination and classify phage contigs in terms of taxonomy and lifestyle. Among the five techniques, we chose the polyethylene glycol concentration method to isolate phages because of its simplicity, low cost, reproducibility, and high number and diversity of phage sequences. 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 depth sequencing on phage diversity and observed the beginning of a plateau for phage contigs at 20,000,000 reads. This work contributes for the development of methods to the isolation of phages in faeces and of their comparative analysis.
The microbiota of the human gut is a complex and rich community where bacteria and their viruses, the phages, are numerically dominant. While the bacterial components have been extensively studied, there are fewer studies on the phage community and no clear standard for isolating the phages and sequencing and analysing their genomes. Since this makes comparisons between studies difficult, we aimed at defining an easy, low-cost, and reproducible methodology for each of these three steps. We analysed five different techniques to isolate phages from human adult faeces and developed a pipeline to analyse their genomes in order to quantify contamination and classify phage contigs in terms of taxonomy and lifestyle. Among the five techniques, we chose the polyethylene glycol concentration method to isolate phages because of its simplicity, low cost, reproducibility, and high number and diversity of phage sequences. 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 depth sequencing on phage diversity and observed the beginning of a plateau for phage contigs at 20,000,000 reads. This work contributes for the development of methods to the isolation of phages in faeces and of their comparative analysis.