Browsing by Author "Dikareva, Evgenia"

The gut microbiota has a major impact on the health and early life development in humans. Viruses infecting prokaryotes, called bacteriophages, are the most abundant group of the gut virome that shapes the prokaryotic community. They have been shown to directly interact with the human host or indirectly by interfering with the gut bacterial community. While in the recent years many studies have explored the human gut virome, the field is currently under active investigation, but no standardised protocols for creating high-throughput virome extractions or bioinformatic pipelines for sequences analyses is available. The first aim of this study was to (1) compare the most promising methods for viral particle concentration (dithiothreitol (DTT) and polyethylene glycol (PEG)), DNA extraction afterwards and scaling the methods for high-throughput procedure. The second aim was to (2) compare four bioinformatics tools: Centrifuge, MetaPhlAn, Gut Virome Database (GVD) and a combination of Centrifuge, MetaPhlAn, VirFinder and Blast (Consensus) by analysing shotgun metagenome sequencing results of infant’s stool samples at three time points: 1, 6 and 12 months. The adjustments for high-throughput DNA extraction, resulted in five protocols. The highest yield of DNA was achieved for 1- and 12-months samples with the PEG method. On the other hand, the DTT method was the best for 6-month samples. The infant’s age was the only significant factor driving the viral composition differences on family level for MetaPhlAn (p = 0.004), Centrifuge (p = 0.001) and Consensus (p = 0.001) methods. However, the number of annotated reads and the virome composition depended exclusive on the software used (p = 0.001). All the methods identified phage families: Siphoviridae, Podoviridae and Myoviridae. GVD was the only method that annotated up to 90% of reads to viruses. In conclusion, our results suggest that the PEG extraction method may be best suited for large-scale virome enrichment, as it allowed to obtain the highest DNA yield, was suitable for high-throughput extractions and allowed to create a virome with a high variability in phage representation. For the novel virus identification, GVD method would be used further as it annotated most of the reads to phages.