Accurate and reproducible whole-genome genotyping for bacterial genomic surveillance with Nanopore sequencing data

Despite recent advances in error rate reduction, until recently, Oxford Nanopore Technologies (ONT) sequences lacked the accuracy required for fine-scale bacterial genomic analysis. Here, recent software improvements of ONT and the ONT-core-genome multilocus sequence typing (cgMLST)-Polisher within the SeqSphere⁺ software were evaluated. We used short-read (Illumina) and long-read ONT sequences of 80 multidrug-resistant organisms (MDROs) for benchmarking. Illumina reads were de novo assembled using SKESA. For ONT, Dorado Super Accurate (SUP) model v.4.3 or v.5.0 basecalled reads were assembled with Flye and then polished with Medaka v.1.12 m4.3 or Medaka v.2.0 bacterial methylation model. In addition, the ONT-cgMLST-Polisher was run over all assemblies. The "ground truth" (GT) hybrid assemblies were created using Hybracter v.0.10.0. Sixteen isolates from four species out of the original 80 isolates were sent to six laboratories for a ring trial. The 80 MDROs basecalled with SUP m4.3 had an average cgMLST allele distance (AD) to the GT of 4.94 with Medaka v.1.12 and 1.78 with Medaka v.2.0, respectively. After further polishing the Medaka v.2.0 data with the ONT-cgMLST-Polisher, the AD dropped to 0.09. Using data basecalled with SUP m5.0 with Medaka v.2.0 further reduced the AD significantly to 0.04. While the ring trial data basecalled with Dorado SUP m4.3 showed more variability and insufficient results for some samples, model 5.0 data resulted in average ADs of 0.36 and 0.17 without and with the ONT-cgMLST-Polisher, respectively. In conclusion, recent ONT Dorado and Medaka models combined with the ONT-cgMLST-Polisher improved ONT sequencing accuracy and made it sufficiently reproducible for genomic surveillance of bacteria.IMPORTANCEONT sequencing methodology is especially attractive for small and medium-sized laboratories due to its relatively low capital investment and price per sample consumable costs. However, until recently, it lacked accuracy and reproducibility for bacterial genomic genotyping. Here, we present an evaluation of the most recent ONT bioinformatic (basecalling and polishing of consensus) improvements and a new ONT-cgMLST-Polisher tool. We demonstrate that by applying those procedures, ONT whole-genome genotyping-based surveillance of bacteria is finally accurate and reproducible enough for routine application even in small laboratories.