Abstract
Microbial genomes can be assembled from short-read sequencing data, but the assembly contiguity of these metagenome-assembled genomes is constrained by repeat elements. Correct assignment of genomic positions of repeats is crucial for understanding the effect of genome structure on genome function. We applied nanopore sequencing and our workflow, named Lathe, which incorporates long-read assembly and short-read error correction, to assemble closed bacterial genomes from complex microbiomes. We validated our approach with a synthetic mixture of 12 bacterial species. Seven genomes were completely assembled into single contigs and three genomes were assembled into four or fewer contigs. Next, we used our methods to analyze metagenomics data from 13 human stool samples. We assembled 20 circular genomes, including genomes of Prevotella copri and a candidate Cibiobacter sp. Despite the decreased nucleotide accuracy compared with alternative sequencing and assembly approaches, our methods improved assembly contiguity, allowing for investigation of the role of repeat elements in microbial function and adaptation.
Highlights
Microbial genomes can be assembled from short-read sequencing data, but the assembly contiguity of these metagenome-assembled genomes is constrained by repeat elements
De novo generation of finished metagenome-assembled genomes (MAGs) for bacteria and archaea is a longstanding goal in microbiome research
The application of long-read methods to analyze gut microbiomes has been hindered by the lack of efficient methods to extract high molecular weight (HMW) DNA from stool
Summary
Microbial genomes can be assembled from short-read sequencing data, but the assembly contiguity of these metagenome-assembled genomes is constrained by repeat elements. We present a workflow for nanopore sequencing of stool samples, including protocols for DNA extraction and genome assembly (Supplementary Fig. 1).
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