Single laboratory evaluation of the (Q20+) nanopore sequencing kit for bacterial outbreak investigations

Abstract This study aimed to evaluate the potential of Oxford Nanopore Technologies (ONT) GridION with Q20+ chemistry as a rapid and accurate method for identifying and clustering foodborne pathogens. The study focuses on assessing whether ONT Q20+ technology could offer near real-time pathogen identification, including SNP differences, serotypes, and antimicrobial resistance genes, to overcome the drawbacks of existing methodologies. This pilot study evaluated different combinations of two DNA extraction methods (Maxwell RSC Cultured Cell DNA kit, and Monarch high molecular weight extraction kits) and two ONT library preparation protocols (ligation and the rapid barcoding sequencing kit) using five well-characterized strains representing diverse foodborne pathogens. The results showed that any combination of extraction and sequencing kits produced high-quality closed bacterial genomes. However, there were variations in assembly length and genome completeness based on different combinations of methods, indicating the need for further optimization. in silico analyses demonstrated that the Q20+ nanopore sequencing chemistry accurately identified species, genotyped, and detected virulence factors comparable to Illumina sequencing. Phylogenomic clustering methods showed that ONT assemblies clustered with reference genomes, although some indels and SNP differences were observed. There were also differences on SNP accuracy among the different species. The observed SNP differences were likely due to sequencing and analysis processes rather than genetic variations in the sampled bacteria. The study also compared a change in the basecaller model with the previous model (SUP 4Khz 260 bps) and found no significant difference in accuracy (SUP 5Khz 400 bps). In conclusion, the evaluation of ONT Q20+ nanopore sequencing chemistry demonstrated its potential as an alternative for rapid and comprehensive bacterial genome analysis in outbreak investigations. However, further research, verification studies, and optimization efforts are needed to address the observed limitations to adopt and fully realize the impact of nanopore sequencing on public health outcomes and more efficient responses to foodborne disease threats.