![]() Specifically, generation of short fragmented assemblies impedes the analysis of genomic context or detection of viral sequences using programs, such as VirSorter 18, 19, which requires long genomic fragments with sufficient evidence to warrant a prediction 20. However, the short-read assembly approaches do have limitations, particularly in assembly contiguity 16, 17. These previous studies, however, were based on short-read sequencing data generated using Illumina sequencer. Such metagenomic signs of coevolution have been missed in previous large viromic studies in humans 3. Re-analysis of the Earth’s virome data revealed signatures of genetic conflict invoked by the coevolution of phages and host oral bacteria enriched in the human oral cavity 15, suggesting it as an attractive system to study coevolution using metagenomic data. More recently, shotgun metagenome sequencing of 3042 samples from various environments, including the human oral cavity, was conducted in a project aimed at uncovering the Earth’s virome 13, which achieved an almost 3-fold increase in the metagenome samples 14. The largest oral virome study was conducted in 2015, which generated and analyzed more than 100 Gb shotgun sequencing data from 25 samples (20 dental plaque specimens and 5 salivary) to primarily explore the phage–bacteria interaction network 12. However, only a small number of metagenomic studies focused on oral phage communities 5, including a study in 2014 that reported an alteration of virome composition in subjects with periodontal disease 11. Attempts have also been made to explore the associations between human gut virome alterations and diseases 7, 9, 10. For example, there have been more attempts to characterize “healthy gut phageome” since a study in 2016 conducted deep-sequencing of DNA from virus-like particles and revealed the presence of completely assembled phage genomes in 64 healthy individuals around the world 8. With the rise of next-generation metagenome sequencing technologies, human gut virome studies have increased rapidly 7. Among them, bacteriophages (phages), or bacterial viruses, in the intestinal microbiome have received increasing attention over the last decade 5, 6, whereas those present in the oral microbiota have been less studied. The two most diverse human microbiomes are intestinal and oral microbiota, which harbor hundreds of coexisting species, including bacteria and viruses. Interspecies networks within the microbiome can modulate energy metabolism pathways and affect human health. Human microbiomes are of enormous interest to researchers 1, 2 and have been model systems for studying polymicrobial communities 3, 4. Our study demonstrates the power of long-read metagenomics utilizing PromethION in uncovering bacteriophages and their interaction with host bacteria. Furthermore, our study suggests that oral phages present in human saliva are under selective pressure to escape CRISPR immunity. Pan-genome analysis of the phages/prophages reveals remarkable diversity, identifying 0.3% and 86.4% of the genes as core and singletons, respectively. ![]() 86% of the phage/prophage group and 67% of the jumbo phages/prophages contain remote homologs of antimicrobial resistance genes. Our analyses also identify a Streptococcus phage/prophage group and nine jumbo phages/prophages. Our analyses demonstrate enhanced scaffolding, and the ability to place a prophage in its host genomic context and enable its taxonomic classification. Our analyses, which integrate both PromethION and HiSeq data of >30 Gb per sample with low human DNA contamination, identify hundreds of viral contigs 0–43.8% and 12.5–56.3% of the confidently predicted phages and prophages, respectively, do not cluster with those reported previously. Here, we conduct a long-read metagenomic study of human saliva using PromethION. Although a few metagenomic studies have focused on oral phages, they relied on short-read sequencing. Bacteriophages (phages), or bacterial viruses, are very diverse and highly abundant worldwide, including as a part of the human microbiomes.
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