Long‐Read Sequencing Reveals Extensive DNA Methylations in Human Gut Phagenome Contributed by Prevalently Phage‐Encoded Methyltransferases

Abstract

DNA methylation plays a crucial role in the survival of bacteriophages (phages), yet the understanding of their genome methylation remains limited. In this study, DNA methylation patterns are analyzed in 8848 metagenome‐assembled high‐quality phages from 104 fecal samples using single‐molecule real‐time sequencing. The results demonstrate that 97.60% of gut phages exhibit methylation, with certain factors correlating with methylation densities. Phages with higher methylation densities appear to have potential viability advantages. Strikingly, more than one‐third of the phages possess their own DNA methyltransferases (MTases). Increased MTase copies are associated with higher genome methylation densities, specific methylation motifs, and elevated prevalence of certain phage groups. Notably, the majority of these MTases share close homology with those encoded by gut bacteria, suggesting their exchange during phage–bacterium interactions. Furthermore, these MTases can be employed to accurately predict phage–host relationships. Overall, the findings indicate the widespread utilization of DNA methylation by gut DNA phages as an evasion mechanism against host defense systems, with a substantial contribution from phage‐encoded MTases.

PacBio sequencing of human fecal samples uncovers the DNA methylation landscape of 22 673 gut phages

“…We found that 6mA methylation rate of temperate phages was higher than that of virulent phages, whereas 4mC methylation rate was the opposite, suggesting that 6mA and 4mC may play different roles in phages of different lifestyles. This result was consistent with previous study ( 38 ). 6mA plays key roles in the regulation of RM system, DNA replication and gene expression, which can affect the survival of temperate phages in the host ( 45 , 46 ).…”

“…A previous study had used Oxford Nanopore technology to detect 5mC and 6mA methylation in five gut viruses with 10× depth ( 14 ). A recent study investigated phage 4mC and 6mA methylation through PacBio sequencing with 412× depth, which may present only a part of the global picture of phage DNA methylation as discussed ( 38 ). In our study, ultra-deep (2120×) PacBio sequencing was used to detect 4mC and 6mA methylation, and the number of detected methylation sites reached a maximum with increased sequencing depth, showing that as many methylation signals as possible with current technology were detected under the ultra-deep PacBio sequencing.…”

MicroWorldOmics: A “One step for All” desktop application for comprehensive analysis in microbiome and virome and exploring “Dark Matter” from MicroWorld

“…The different modules are extracted from the generated tree using hierarchical clustering (dynamicTreeCut) [36]. (5). Differential network visualization.…”

“…The advances in high-throughput sequencing technologies have provided a new perspective to microbial world analysis at a global scale, for instance, based on deep metagenomic sequencing, the evolutionary dynamics of multiethnic reproductive tract microbiome have been revealed [2], Genome-resolved metagenomics of environmental samples allows the discovery of new biosynthetic gene clusters [3]. The construction of the Unified Ruminant Phage Catalogue (URPC) [4] and Chinese Human Gut Virome (CHGV) [5] is helpful in understanding the process of gene exchange between phage and host bacteria and also provides a highprecision data set for the confirmation of phage-host relationship. With the 'microworld' field being paid more attention and more related data generated, methods and tools for integrating data analysis need to and the initial files could be uploaded to a remote server for analysis, such as Genomad [19] (https://github.com/apcamargo/genomad/) and Prokka [20] (https://github.com/tseemann/prokka).…”

Gut virome in inflammatory bowel disease and beyond

“…Recently, single-virus sequencing has emerged as a promising tool for uncovering the diversity and abundance of viruses 46 47 and for the identification of host–phage interactions. 48 Coupled with third-generation long-read sequencing, 49 single-virus sequencing may serve as a powerful framework for the accurate recovery of viral genomes and high-throughput cataloguing of host–phage interactions. Both will facilitate the formation of phage cocktails targeting IBD-related bacteria.…”