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

Abstract

DNA sequencing and other DNA-based methods are now broadly used for detection and identification of bacterial foodborne pathogens. For the identification of foodborne bacterial pathogens, taxonomic assignments must be made to the species or even subspecies level. Long-read DNA sequencing provides finer taxonomic resolution than short-read sequencing. Here, we demonstrate the potential of long-read shotgun sequencing obtained from the Oxford Nanopore Technologies (ONT) MinION single-molecule sequencer, in combination with the Basic Local Alignment Search Tool (BLAST) with custom sequence databases, for foodborne pathogen identification. A library of mixed DNA from strains of the "Super-7" Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, O145, and O157[:H7]) was sequenced using the ONT MinION resulting in 44,245 long-read sequences. The ONT MinION sequences were compared to a custom database composed of the E. coli O-antigen gene clusters. A vast majority of the sequence reads were from outside of the O-antigen cluster and did not align to any sequences in the O-antigen database. However, 58 sequences (0.13% of the total sequence reads) did align to a specific Super-7 O-antigen gene cluster, with each O-antigen cluster aligning to at least four sequence reads. BLAST analysis against a custom whole-genome database revealed that 5096 (11.5%) of the MinION sequence reads aligned to one and only one sequence in the database, of which 99.6% aligned to a 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.