Long-read sequencing reveals extensive DNA methylations in human gut phagenome contributed by prevalently phage-encoded methyltransferases

Abstract DNA methylation is essential for the survival of bacteriophages (phages), yet, we know little about the methylation of their genomes. Here, we analyze the DNA methylation patterns of 8,848 metagenome-assembled high-quality phages across 104 fecal samples using single-molecule real-time (SMRT) sequencing. We show that 97.60% of gut phages could be methylated, and reveal factors that correlate with methylation densities. Phages having higher methylation densities are more prevent, suggesting putative viability advantages of the DNA methylation. Strikingly, more than a third of the phages encode their own DNA methyltransferases (MTases). More MTase copies are associated with increased genome methylation densities, methylation motifs, and higher prevalence of certain phage groups. Most MTases are closely homologous to gut bacterium-encoded ones, likely exchanged during phage-bacterium interactions, and could be used to accurately predict phage-host relationships. Taken together, our results suggest that the gut DNA phages universally use DNA methylation to escape from host defense systems with significant contribution from phage-encoded MTases.