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 program, 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; Autocycler), and three hybrid assemblers (Hybracter hybrid; Unicycler normal; 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 between 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 SNPs and 0 IQR:0-1 indels per genome; quality value/Q score, 100 IQR: 64-100), with only 4/92 genome sequences having >10 SNPs/indels. Medaka polishing with un-subsampled long-reads resulted in small improvements in indels but not SNPs for both Flye and Autocycler assemblies. Seven-locus MLST, 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.