Long-read shotgun metagenome sequencing using PromethION uncovers novel bacteriophages, their abundance, and interaction with host bacterial immunity in the oral microbiota

Abstract

17 18 Bacteriophages (phages), or bacterial viruses, are very diverse and highly abundant worldwide, 19 including human microbiomes. Although a few metagenomic studies have focused on oral 20 phages, they relied on short-read sequencing. Here, we conducted a long-read metagenomic 21 study of human saliva for the first time using PromethION that requires a smaller amount of 22 DNA than PacBio. Our analyses, which integrated both PromethION and HiSeq data of >30 Gb 23 per sample, revealed N50 ranging from 187-345 kb and thousands of contigs with >1 kb 24 accounting for > 99% of all contigs on which 94-96% of HiSeq reads were mapped. We 25 identified hundreds of viral contigs (95 phages and 333 prophages on an average per sample); 26 0-43.8% and 12.5-56.3% of the "most confident" phages and prophages, respectively, didn't 27 cluster with those reported previously and were identified as novel. Our integrated analyses 28 identified highly abundant oral phages/prophages, including a novel Streptococcus phage 29 cluster and nine jumbo phages/prophages. Interestingly, 86% of the phage cluster and 67% of 30 the jumbo phages/prophages contained remote homologs of antimicrobial resistance genes, 31 suggesting their potential role as a source of recombination to generate new resistance genes. 32Pan-genome analysis of the phages/prophages revealed remarkable diversity, identifying 0.3% 33 and 86.4% of the genes as core and singletons, respectively. Functional annotation revealed 34 that the highest fraction of the core genes was enriched in phage morphogenesis, followed by 35 the fraction enriched in host cellular processes. Furthermore, our study suggested that oral 36 phages present in human saliva are under selective pressure for escaping CRISPR immunity. 37 (250/250 words) 38 39 Importance 40 41Despite the abundance and grave implications oral bacterial viruses in health and disease, little 42 is known regarding the different groups of oral bacterial viruses, their relative abundances 43 under various conditions, and their activities. We provided answers to these questions for the first time utilizing a recently developed sequencer that can capture and sequence long DNA 45 fragments, including viruses, and requires only a small amount of DNA input, making it 46 suitable for analyzing human oral samples. We identified hundreds of viral sequences, 47 (147/150 words) 54 55

Hidden viral sequences in public sequencing data and warning for future emerging diseases

“…3B and 3C ). Further improvement in sequence assembly efficiency (40) or integrative analysis using short- and long-read sequence datasets (41) can solve this problem. Second, there may be a bias in virus detection using public sequencing data depending on their genomic types.…”