Rapid whole genome and plasmid sequencing of foodborne pathogens

In the US alone approximately 1 in 6 people are affected by foodborne pathogens each year, resulting in an estimated 3,000 deaths. Public health agencies typically perform outbreak tracking and surveillance during food production using short-read sequencing technologies; however, these techniques are expensive, time consuming, and limited to laboratory settings. In addition, the inherent read length limitations of traditional sequencing technologies results in the generation of fragmented genome assemblies, limiting the potential to determine co-regulated or co-transmissible genes associated with mobile genetic elements.

Identifying nanopore sequencing as a potential solution to these challenges, Taylor et al.¹ used the MinION to sequence two bacterial strains, Salmonella enterica subsp. enterica serovar Bareilly and Escherichia coli O157:H7, associated with previous foodborne pathogen outbreaks. Purified genomic DNA from the two culture samples was prepared using the Ligation Sequencing Kit and barcoded using the Native Barcoding Kit prior to sequencing on a single MinION Flow Cell. A customised data analysis workflow comprising publicly available tools (including Unicycler and nanopolish) was used to generate de novo genome assemblies.

A total of 2.8 billion and 3.8 billion bases corresponding to a mean sequencing depth of 599x and 692x were generated for S. enterica and E. coli respectively. The longest read obtained was 129 kb in length.

To assess the effect of sequencing run time on the final assembly quality, the data for both isolates were subsampled at various time intervals — from 15 minutes to 960 minutes. Four hours (240 mins) was determined as the shortest run time sufficient to assemble circular sequences for all chromosomes and plasmids of both isolates. Based on these results, the authors suggest that six bacterial samples could be multiplexed on a single MinION Flow Cell, with complete, highly accurate genomic data generation within 16 hours.

‘Using MinION sequencing alone, two completely closed contigs, one chromosome and one plasmid for each pathogen, were assembled’ ¹

Comparing these nanopore-only assemblies against their respective reference genomes gave an average nucleotide identity of 99.87% for S. enterica and 99.89% for E. coli. Annotation of the assemblies using Geneious confirmed the accuracy of the genomes, identifying serotyping antigens, virulence factors, and AMR genes (Figure 1).

Figure 1: Nanopore sequencing enabled, complete and accurate genome and plasmid assembly. (a) The E. coli genome annotation identified 5,748 coding sequences (CDS), 106 tRNAs, 29 rRNAs, 6 regulatory regions (green), and 1 repeat region (brown). The CDS for two virulence factors, LEE and Shiga toxin subunits are highlighted in yellow. (b) The E. coli pO157 annotation shows all 124 coding sequences (CDS) in yellow. The CDS of three wellknown virulence factors are highlighted: haemolysin (ehx), catalaseperoxidase (katP), and the type II secretion system (T2SS). Image from Taylor et al. and available under Creative Commons license (creativecommons. org/licenses/by/4.0) ¹.

Furthermore, the team also demonstrated that the nanopore data obtained after just four hours of sequencing allowed the generation of phylogenetic trees which were consistent with those obtained using short-read sequencing technology. Summarising this research, the authors suggest that ‘this low-cost, rapid, random-priming nanopore sequencing approach, coupled with our customized workflow, provides sufficient data where complete genomes, including plasmids, can be assembled into a single contiguous sequence with 99.89% accuracy’ ¹.

‘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’ ¹