Abstract
BackgroundDespite the relevance of viral populations, our knowledge of (bacterio) phage populations, i.e., the phageome, suffers from the absence of a “gold standard” protocol for viral DNA extraction with associated in silico sequence processing analyses. To overcome this apparent hiatus, we present here a comprehensive performance evaluation of various protocols and propose an optimized pipeline that covers DNA extraction, sequencing, and bioinformatic analysis of phageome data.ResultsFive widely used protocols for viral DNA extraction from fecal samples were tested for their performance in removal of non-viral DNA. Moreover, we developed a novel bioinformatic platform, METAnnotatorX, for metagenomic dataset analysis. This in silico tool facilitates a range of read- and assembly-based analyses, including taxonomic profiling using an iterative multi-database pipeline, classification of contigs at genus and species level, as well as functional characterizations of reads and assembled data. Performances of METAnnotatorX were assessed through investigation of seven mother-newborn pairs, leading to the identification of shared phage genotypes, of which two were genomically decoded and characterized.METAnnotatorX was furthermore employed to evaluate a protocol for the identification of contaminant non-viral DNA in sequenced datasets and was exploited to determine the amount of metagenomic data needed for robust evaluation of human adult-derived (fecal) phageomes.ConclusionsResults obtained in this study demonstrate that a comprehensive pipeline for analysis of phageomes will be pivotal for future explorations of the ecology of phages in the gut environment as well as for understanding their impact on the physiology and bacterial community kinetics as players of dysbiosis and homeostasis in the gut microbiota.
Highlights
Despite the relevance of viral populations, our knowledge of phage populations, i.e., the phageome, suffers from the absence of a “gold standard” protocol for viral DNA extraction with associated in silico sequence processing analyses
Comparative evaluation of various protocols for viral DNA extraction and purification Virome protocol analyses commonly consist of the isolation of virus-like particles (VLPs) from a fecal sample followed by extraction of the genetic material from these Virus-like particle (VLP), prior to further analysis of the obtained genetic material by means of shotgun sequencing approaches [21,22,23]
It was determined through a phage spiking experiment that PEG removal by buffer exchange was inefficient and caused loss of phages during subsequent centrifugation; DNA extraction was directly performed on the resuspended PEG-precipitated VLPs
Summary
Despite the relevance of viral populations, our knowledge of (bacterio) phage populations, i.e., the phageome, suffers from the absence of a “gold standard” protocol for viral DNA extraction with associated in silico sequence processing analyses. This rather naïve view of phage ecology in the GIT reflects the very limited exploration of the role, if any, of phages in the development and evolution of common gut diseases, with studies focusing mainly on inflammatory bowel diseases (IBD), such as Crohn’s disease (CD) and ulcerative colitis (UC) [13, 14] This knowledge gap can primarily be attributed to the lack of a comprehensive experimental pipeline for metagenomic analyses of viral populations that ideally should include an efficient and reliable protocol for viral DNA extraction and purification, as well as bioinformatic tools for phageome data management, processing and associated analysis. These limitations underline the need for a thorough assessment of available methodologies for phageome analysis, with particular focus on the identification of the viral DNA extraction protocol providing the lowest relative abundance of exogenous DNA, as well as definition of a comprehensive bioinformatic pipeline for phylogenetic and genomic characterization of the viral population
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
