Rapid, multiplexed, whole genome and plasmid sequencing of foodborne pathogens using long-read nanopore technology

<jats:title>Abstract</jats:title><jats:p>U.S. public health agencies have employed next-generation sequencing (NGS) as a tool to quickly identify foodborne pathogens during outbreaks. Although established short-read NGS technologies are known to provide highly accurate data, long-read sequencing is still needed to resolve highly-repetitive genomic regions and genomic arrangement, and to close the sequences of bacterial chromosomes and plasmids. Here, we report the use of long-read nanopore sequencing to simultaneously sequence the entire chromosome and plasmid of <jats:italic>Salmonella enterica subsp. enterica</jats:italic> serovar Bareilly and <jats:italic>Escherichia coli</jats:italic> O157:H7. We developed a rapid and random sequencing approach coupled with <jats:italic>de novo</jats:italic> genome assembly within a customized data analysis workflow that uses publicly-available tools. In sequencing runs as short as four hours, using the MinION instrument, we obtained full-length genomes with an average identity of 99.87% for <jats:italic>Salmonella</jats:italic> Bareilly and 99.89% for <jats:italic>E. coli</jats:italic> in comparison to the respective MiSeq references. These nanopore-only assemblies provided readily available information on serotype, virulence factors, and antimicrobial resistance genes. We also demonstrate the potential of nanopore sequencing assemblies for rapid preliminary phylogenetic inference. Nanopore sequencing provides additional advantages as very low capital investment and footprint, and shorter (10 hours library preparation and sequencing) turnaround time compared to other NGS technologies.</jats:p>