Complete, closed bacterial genomes from microbiomes using nanopore sequencing

TLDR

Microbial genomes can be assembled from short‑read sequencing, but repeat elements limit contiguity, making accurate placement of repeats essential for understanding genome structure and function. The study aimed to generate closed bacterial genomes from complex microbiomes using nanopore sequencing. We used the Lathe workflow, integrating long‑read assembly and short‑read error correction, validated on a synthetic 12‑species mixture and applied to 13 human stool samples to produce closed genomes. Seven genomes were assembled into single contigs, three into four or fewer contigs, and 20 circular genomes—including *Prevotella copri* and a candidate *Cibiobacter* sp.—were recovered from human stool, demonstrating that despite lower nucleotide accuracy, the approach improves contiguity and enables study of repeat elements in microbial function and adaptation.

Abstract

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.