Long-read Nanopore sequencing reveals multiple modes of capsid-mediated mobilisation of bacterial genomic DNA

Here, we present a novel approach to study packaging and mobilisation of bacterial DNA in complex microbial communities, such as the human gut microbiome, by viral or virus-like capsids.Defective bacteriophage (phage) particles and gene transfer agents (GTAs) are constrained by their capsids’ structural properties in the length of host DNA they can package. This results in a narrow distribution of DNA fragment lengths associated with a given viral carrier. Oxford Nanopore long read sequencing of intact capsid-packaged single DNA molecules can be used to measure their lengths and identify read lengths peaks associated with specific viral carrier agents. The methodology developed in this study was validated using a selection of well-characterised transducing phages and a GTA-like element, and then applied to study bacterial DNA encapsidation and mobilisation in the faecal microbiomes from three healthy human donors.Bacterial DNA encapsidation and mobilisation appears to be widespread in the microbiome with up to 4% of capsid-packaged DNA in he gut virome being of bacterial (non-prophage) origin. Generalised transduction and GTA activity is especially prevalent in the family Ospillospiraceae (genera Faecalibacterium, Gemmiger, Onthomonas), whereas examples of lateral transduction were observed in genus Bacteroides. In addition to that induction of prophages in a variety of highly prevalent anaerobic bacteria was observed.Further work will be needed to understand biological significance of capsid mediated DNA packaging in the gut microbiome and its role in community structure, evolution on ecological timescales, spread of beneficial and detrimental genes, as well as its possible role in gastrointestinal disease.R_scripts_and_input_data.tgz - complete set of R scripts and input data needed to recreate all tables and figures in the manuscriptlong_reads_length_and_mapping_figs.zip - complete set of raw figures describing read length distributions and genomic scaffold mapping patterns of long Nanopore reads (used to perform manual analysis and picking of VLP packaging events described in the manuscript)MAGs_scaffolds_reads.zip - MAGs, scaffolds, long reads, short read coverage as .csv and .RDS files