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

Authors: Taylor, Tonya; VOLKENING, JEREMY - Base2bio; DEJESUS, ERIC - Food Safety Inspection Service (FSIS); SIMMONS, MUSTAFA - Food Safety Inspection Service (FSIS); DIMITROV, KIRIL - Consultant; GLENN, TILLMAN - Food Safety Inspection Service (FSIS); Suarez, David; Afonso, Claudio

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/14/2019
Publication Date: 11/8/2019
Citation: Taylor, T.L., Volkening, J.D., Dejesus, E., Simmons, M., Dimitrov, K.M., Glenn, T.E., Suarez, D.L., Afonso, C.L. 2019. Rapid, multiplexed, whole genome and plasmid sequencing of foodborne pathogens using long-read nanopore technology. Scientific Reports. 9:16350. https://doi.org/10.1038/s41598-019-52424-x.
DOI: https://doi.org/10.1038/s41598-019-52424-x

Interpretive Summary: In the U.S., approximately one of six individuals are sickened by foodborne illnesses, which results in approximately 3,000 deaths each year. In order to quickly detect and identify the source of the contamination, many agencies have employed next-generation sequencing (NGS) in response to an outbreak. However, the available short-read technologies are sometimes not sufficient in resolving large repetitive regions of bacterial genomes or sequencing complete antibiotic or virulence plasmids, and therefore, in the past, have had to be paired with an expensive, long-read technology. The significance of our study is in demonstrating that the recently developed long-read technology from Oxford Nanopore, paired with our customized workflow that employs publicly available tools, can independently sequence and assemble the complete chromosome and plasmid of two of the most prominent foodborne pathogens. The obtained data provided readily available information on serotype, virulence factors, and antibiotic resistance genes, and the consensus sequences had 99.87% and 99.89% nucleotide identity to their respective reference sequences.

Technical Abstract: 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 Salmonella enterica subsp. enterica serovar Bareilly and Escherichia coli O157:H7. We developed a rapid and random sequencing approach coupled with de novo 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 Salmonella Bareilly and 99.89% for E. coli 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.