Accurate and Reproducible Whole-Genome Genotyping for Bacterial Genomic Surveillance with Nanopore Sequencing Data
Abstract Despite recent advances in error rate reduction, until recently Oxford Nanopore Technology (ONT) sequences lacked the accuracy required for fine scale bacterial genomic analysis. Here, recent software improvements of ONT and the ONT-cgMLST-Polisher within the SeqSphere+ software were evaluated. We used short- (Illumina) and long-read ONT sequences of 80 multidrug-resistant bacteria (MDROs) for benchmarking. Illumina reads were de-novo assembled with SKESA. For ONT, Dorado super accurate (SUP) model 4.3 or 5.0 basecalled reads were assembled with Flye and then polished with Medaka v1.12 m4.3 or Medaka v2.0 bacterial methylation model. In addition, the ONT-cgMLST-Polisher was run over all assemblies. The 'ground truth' (GT) hybrid assemblies were created using Hybracter v0.10.0. 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 v1.12 and 1.78 with Medaka v2.0, respectively. After further polishing the Medaka v2.0 data with the ONT-cgMLST-Polisher, the AD dropped to 0.09. Using data basecalled with SUP m5.0 with Medaka v2.0 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.