Complete, closed bacterial genomes from microbiomes using nanopore sequencing

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.

Fig. 1. Taxonomic read composition, per-organism read-length distributions and genome assemblies in a defined 12-species bacterial mixture.

a, Relative read counts are shown for the expected equal composition of bacterial cells and the observed composition, with correction for relative genome size. b, Read-length distributions per organism. Individual organisms demonstrate varying read-length distributions in some cases. c, Circos plots demonstrate the relative assembly contiguity of the nanopore versus short-read assembly approaches. Nanopore sequencing and assembly (colored outer ring) outperforms short-read assembly (black inner ring), producing complete genome assemblies (small black inner dots) in seven of 12 cases, with a further three assembled in four contigs or fewer. Numbers indicate genome size in megabases. Note that complete assemblies may contain one apparent break due to differing linearization breakpoints in reference and assembly sequences.

Fig. 2. Per-organism assembly contiguity, diversity and taxonomic read composition in two healthy human stool microbiomes.

a, Species-level Shannon diversity is shown for the sequence datasets obtained. Higher diversity is found in libraries prepared with the present DNA extraction method. Relative species-level abundances are shown for a conventional workflow consisting of bead-beating extraction and short-read sequencing, as well as the present workflow consisting of HMW DNA extraction and long-read sequencing. b, Contiguity is expressed as per-bin N50 divided by per-bin length (the total length of sequences assigned to the bin). As bin assembly approaches completion, the quantity N50 divided by bin length approaches one, regardless of genome size. Nanopore sequencing and assembly (blue, purple) demonstrates higher assembly contiguity than read-cloud (gold) and short-read (green) approaches. For all organisms achieving assembly N50 of at least 500 kbp or a complete genome draft by any approach, genome draft quality and contiguity are shown for long reads, read clouds and short reads. Shapes indicate draft quality. Asterisk marks a genome later annotated as putative Cibiobacter.

Fig. 3. Circos diagrams of closed, circular genomes of P. copri and Cibiobacter sp.

a, P. copri from P2-A. b, Cibiobacter sp. from P1. In both plots, the outermost ring represents the complete, closed and circularized genome of the given organism. The middle and inner rings represent contigs from the corresponding read-cloud and short-read assemblies, respectively, that were mapped to the nanopore assembly. The inner track in each case displays annotated, predicted mobile genetic elements such as insertion sequences (ISs), transposases and prophage.