Nanopore long-read-only genome assembly of clinical Enterobacterales isolates is complete and accurate

Whole bacterial genome sequence reconstruction using Oxford Nanopore Technologies (‘Nanopore’) long-read-only sequencing may offer a lower-cost, higher-throughput alternative for pathogen surveillance to ‘hybrid’ assembly with recent improvements in Nanopore sequencing accuracy. We evaluated the accuracy, including plasmid reconstruction, of Nanopore long-read-only genome assemblies of Enterobacterales. We sequenced 92 genomes from clinical Enterobacterales isolates, collected in England under a national surveillance programme, with long-read Nanopore (R10.4.1, Dorado v5.0.0 super-high-accuracy basecalled) and short-read Illumina (NovaSeq) sequencing approaches. Genomes were assembled using three long-read-only (Flye, Hybracter long and Autocycler) and three hybrid assemblers (Hybracter hybrid, Unicycler normal and bold). Three polishing modalities (Medaka v2 with subsampled or un-subsampled long-reads; Polypolish+Pypolca with short-reads) were investigated. Autocycler circularised the most chromosomes [87/92 (95%)]. Plasmid sequence reconstruction was comparable among all assemblers except Flye, all recovering 90–96% of plasmids, although the ‘ground truth’ was uncertain. Flye performed worse than other assemblers on almost all metrics. Autocycler+Medaka (un-subsampled long-reads) was the most accurate long-read-only assembler/polisher combination, comparable to hybrid assemblies [median 0 (IQR: 0–0) single nucleotide variants (SNVs) and 0 (IQR: 0–1) insertions/deletions (indels) per genome; median quality value/ Q score 100 (IQR: 64–100)], with only 4/92 genome sequences having >10 SNVs/indels. Medaka polishing with un-subsampled long-reads resulted in small improvements in indels, but not SNVs for both Flye and Autocycler assemblies. Seven-locus multi-locus sequence type, antimicrobial resistance, virulence and stress gene annotation was equivalent across assembler/polisher combinations. Nanopore long-read-only bacterial genome assembly with Autocycler combined with Medaka polishing (using un-subsampled reads) is similarly accurate and possibly more complete than hybrid assemblies, representing a viable alternative for incorporating high-quality genomic data, including plasmids, into Enterobacterales surveillance.