Nanopore sequencing enables high-resolution analysis of resistance determinants and mobile elements in the human gut microbiome

Abstract

The analysis of information rich whole-metagenome datasets acquired from complex microbial communities is often restricted by the fragmented nature of assembly from short-read sequencing. The availability of long-reads from third-generation sequencing technologies (e.g. PacBio or Oxford Nanopore) can help improve assembly quality in principle, but high error rates and low throughput have limited their application in metagenomics. In this work, we describe the first hybrid metagenomic assembler which combines the advantages of short and long-read technologies, providing an order of magnitude improvement in contiguity compared to short read assemblies, and high base-pair level accuracy. The proposed approach (OPERA-MS) integrates a novel assembly-based metagenome clustering technique with an exact scaffolding algorithm that can efficiently assemble repeat rich sequences. Based on evaluations with defined in vitro communities and virtual gut microbiomes, we show that it is possible to assemble near complete genomes from metagenomes with as little as 9x long read coverage, thus enabling high quality assembly of lowly abundant species (<1%). Furthermore, OPERA-MSs fine-grained clustering is able to deconvolute and assemble multiple genomes of the same species in a single sample, allowing us to study the complex dynamics of the human microbiome at the sub-species level. Applying nanopore sequencing to gut metagenomes of patients undergoing antibiotic treatment, we show that long reads can be obtained from stool samples in clinical studies to produce more meaningful metagenomic assemblies (up to 200x improvement over short-read assemblies), including the closed assembly of >80 putative plasmid/phage sequences and a 263kbp jumbo phage. Our results highlight that high-quality hybrid assemblies provide an unprecedented view of the gut resistome in these patients, including strain dynamics and identification of novel plasmid sequences.