Evaluation of Oxford Nanopore Technologies workflows for genomic epidemiology of outbreak‐associated bacterial isolates in the clinical setting

Abstract Accurate and efficient whole-genome sequencing (WGS) is crucial for clinical diagnostics and surveillance of bacterial infections. Here, we investigate the potential of a new Oxford Nanopore Technologies (ONT) workflow for WGS of clinically relevant bacterial isolates. Specifically, we assess the performance of R10.4.1 flow cells in combination with the V14 version of the transposase-based (RBK) library preparation kit to provide rapid and accurate genomic epidemiological comparisons of bacterial species of clinical importance. We focused on retrospective collections of outbreak-associated Corynebacterium diphtheriae (CDIP) and vancomycin-resistant Enterococci (VRE), and benchmarked expected performance parameters such as genome assembly quality, genotyping (MLST, cgMLST), SNP profiling and antimicrobial resistance and virulence prediction, against WGS data obtained routinely by Illumina MiSeq sequencing. Complete concordance with Illumina results was observed for MLST in both species, and for cgMLST in CDIP, across all ONT kits and software evaluated. For VRE, however, cgMLST results varied with strain identity, library preparation kit, and analysis parameters, likely due to software challenges to correctly call methylated bases. Yet, the use of the latest basecalling models combined with PCR-based library preparation kit (RPB) reliably reproduced Illumina cgMLST results across all tested VRE strains. By testing two hybrid strategies combining PCR-free and PCR-based library preparation approaches, we also showed that combining PCR-free and PCR-based methods may yield a promising strategy, achieving both high accuracy and assembly completeness. Genomic-based AMR prediction was consistent across sequencing methods, and we further highlight advantages and limitations of the PCR-based, PCR-free, and mixed assemblies, to inform on the genomic context of AMR genes. This study demonstrates that a Nanopore-only sequencing approach may offer improved accuracy and consistency for classical bacterial typing in outbreak investigations, paving the way to wider use in clinical microbiology laboratories. Competing Interest Statement The authors have declared no competing interest. Funding Statement This study did not receive any funding. Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All CDIP and VRE isolates in the present study have been anonymized and no patient information is used in the interpretation of the bacterial genomic data. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data Availability Genome assemblies, Illumina MiSeq and ONT data are available from BioProject PRJNA889706 (http://www.ncbi.nlm.nih.gov/bioproject/889706), and BioProject PRJNA1230056 (http://www.ncbi.nlm.nih.gov/bioproject/1230056). The correction script (Figure 2) is available at: https://github.com/RametteLab/NanoporeHybridKP http://www.ncbi.nlm.nih.gov/bioproject/889706