Evaluation of the accuracy of bacterial genome reconstruction with Oxford Nanopore R10.4.1 long-read-only sequencing

Whole genome reconstruction of bacterial pathogens has become an import tool for tracking antimicrobial resistance spread, however accurate and complete assemblies have only been achievable using hybrid long and short-read sequencing. We have previously found the Oxford Nanopore Technologies (ONT) R10.4/kit12 flowcells produced improved assemblies over the R9.4.1/kit10, however they contained too many errors compared to hybrid Illumina-ONT assemblies. ONT have since released the R10.4.1/kit12 flowcells that promises greater accuracy and yield. They have also released newly trained basecallers using native bacterial DNA containing methylation sites intended to fix systematic errors, specifically Adenosine (A) to Guanine (G) and Cytosine (C) to Thymine (T) substitutions. ONT have recommended the use of Bovine Serum Albumin (BSA) during library preparation to improve sequencing yield and accuracy. To evaluate these improvements, we sequenced DNA extracts from four commonly studied bacterial pathogens, namely Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus, as well as 12 disparate E. coli clinical samples from different phylogroups and sequence types. These were all sequenced with and without BSA. These sequences were de novo assembled and compared against Illumina-corrected reference genomes. Here we have found the nanopore long read-only R10.4.1 (kit14) assemblies with basecallers trained using native bacterial methylated DNA produce accurate assemblies from 40x depth or higher, sufficient to be cost-effective compared to hybrid long-read (ONT) and short-read (Illumina) sequencing. ### Competing Interest Statement The authors have declared no competing interest.