Evaluation of Nanopore sequencing technology to differentiateSalmonellaserotypes and serotype variants with the same or closely related antigenic formulae

ABSTRACT Our previous study demonstrated that whole genome sequencing (WGS) data generated by Oxford Nanopore Technologies (ONT) can be used for rapid and accurate prediction of Salmnonella serotypes. However, one limitation is that established methods for WGS-based serotype prediction cannot differentiate certain serotypes and serotype variants with the same or closely related antigenic formulae. This study aimed to evaluate Nanopore sequencing and corresponding data analysis for differentiation of these serotypes and serotype variants, thus overcoming this limitation. Five workflows that combined different flow cells, library construction methods and basecaller models were evaluated and compared. The workflow that consisted of the R9 flow cell, rapid sequencing library construction kit and guppy basecaller with base modified model performed best for Single Nucleotide Polymorphism (SNP) analysis. With this workflow, as high as 99.98% matched the identity of the assembled genomes and only less than five high quality SNPs (hqSNPs) between ONT and Illumina sequencing data were achieved. SNP typing allowed differentiation of Choleraesuis sensu stricto , Choleraesuis var. Kunzendorf, Choleraesuis var. Decatur, Paratyphi C, and Typhisuis that share the same antigenic formula 6,7:c:1,5. Prophage prediction further distinguished Orion var. 15 + and Orion var. 15 + , 34 + . Our study improves the readiness of ONT as a Salmonella subtyping and source tracking tool for food industry applications. Highlights Salmonella serotypes or serotype variants with the same antigenic formula were differentiated by SNP typing. Nanopore sequencing followed by phage prediction identified the Salmonella serotype variants caused by phage conversion. The latest ONT technology is capable of high fidelity SNP typing of Salmonella .