Serogroup-level resolution of the “Super-7” Shiga toxin-producing Escherichia coli using nanopore single-molecule DNA sequencing

Authors: Peritz, Adam; Chen, Chinyi; Paoli, George; Gehring, Andrew

Submitted to: Analytical and Bioanalytical Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/24/2018
Publication Date: 1/27/2018
Citation: Peritz, A., Chen, C., Paoli, G., Gehring, A.G. 2018. Serogroup-level resolution of the “Super-7” Shiga toxin-producing Escherichia coli using nanopore single-molecule DNA sequencing. Analytical and Bioanalytical Chemistry. https://doi.org/10.1007/s00216-018-0877-1.

Interpretive Summary: In order to prevent the distribution of contaminated foods and reduce the burden of foodborne illness, food producers and regulatory agencies need rapid, accurate and cost-effective methods for the identification of bacterial foodborne pathogens. In the past few years, bacterial genome sequencing (i.e., determining the sequence of a bacterium’s complete set of DNA) has been broadly adopted by regulatory and public health agencies to characterize bacterial pathogens and track outbreaks of foodborne illness. Nevertheless, due to expense and technical limitations, these genomic technologies have not been adopted for rapid foodborne pathogen detection and identification. Recently, an inexpensive and portable DNA sequencing device, the Oxford Nanopore MinION DNA sequencer, which overcomes several of these limitations, was introduced. Here we report a proof-of-concept study that demonstrates the potential application of the MinION sequencer for the inexpensive, rapid, specific, and field-portable detection and identification of foodborne bacterial pathogens.

Technical Abstract: DNA sequencing and other DNA-based methods, such as PCR, are now broadly used for detection and identification of bacterial foodborne pathogens. For the identification of foodborne bacterial pathogens, it is important to make taxonomic assignments to the species, or even subspecies level. Long-read DNA sequencing techniques can result in finer taxonomic resolution of microbes and microbial communities compared to short-read sequencing platforms. In this study we demonstrate the potential for using a combination of long read shotgun sequencing obtained from the Oxford Nanopore Technologies (ONT) MinION single-molecule sequencer and the Basic Local Alignment Search Tool (BLAST) with custom sequence databases for foodborne pathogen identification. A library of DNA extracted from strains of the “Super-7” Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, O145, and O157) was sequenced using the ONT MinION and analyzed using BLAST. Results showed that, despite extensive sequence identity within the STEC core genome, 11.5% of the MinION reads were aligned to a sequence present in only one strain in the database, and 99.6% of these strain-specific DNA sequence reads aligned to a DNA sequence from a “Super-7” STEC. These results demonstrate the ability of the method to resolve STEC to the serogroup level and the potential general utility of the MinION for the detection and typing of foodborne pathogens.