Complete Whole Genome Sequences of Escherichia coli Surrogate Strains and Comparison of Sequence Methods with Application to the Food Industry

Abstract

In 2013, the U.S. Department of Agriculture Food Safety and Inspection Service (USDA-FSIS) began transitioning to whole genome sequencing (WGS) for foodborne disease outbreak- and recall-associated isolate identification of select bacterial species. While WGS offers greater precision, certain hurdles must be overcome before widespread application within the food industry is plausible. Challenges include diversity of sequencing platform outputs and lack of standardized bioinformatics workflows for data analyses. We sequenced DNA from USDA-FSIS approved, non-pathogenic E. coli surrogates and a derivative group of rifampicin-resistant mutants (rifR) via both Oxford Nanopore MinION and Illumina MiSeq platforms to generate and annotate complete genomes. Genome sequences from each clone were assembled separately so long-read, short-read, and combined sequence assemblies could be directly compared. The combined sequence data approach provides more accurate completed genomes. The genomes from these isolates were verified to lack functional key E. coli elements commonly associated with pathogenesis. Genetic alterations known to confer rifR were also identified. As the food industry adopts WGS within its food safety programs, these data provide completed genomes for commonly used surrogate strains, with a direct comparison of sequence platforms and assembly strategies relevant to research/testing workflows applicable for both processors and regulators.

Long-Read Sequencing and Hybrid Assembly for Genomic Analysis of Clinical Brucella melitensis Isolates

“…While there were instances of gaps in the short-read assembly and errors in long-read assemblies, this did not ultimately affect identification of virulence genes. These findings are similar to work investigating Illumina vs. ONP sequencing with Escherichia coli surrogate strain isolates [ 58 ]. In general, long-read sequencing had notable limitations in regard to phylogenetic analysis, and long-read assemblies generally failed to cluster closely enough with their short-read and hybrid counterparts to be considered as epidemiologically related based on a previously established threshold.…”

Molecular detection and characterization of foodborne bacteria: Recent progresses and remaining challenges

“…While hybrid assembly's approaches were found to gen-TA B L E 3 Description of several sequencing platforms commonly used for whole genome sequencing. This table was updated from Jagadeesan et al (2019) erate fragmentation and accuracy biases based upon the initial assembly strategy, they delivered promising results in terms of genome coverage for several pathogenic bacteria relevant to the food industry (Chen, Kuang et al, 2020;Commichaux et al, 2021;Neal-McKinney et al, 2021;Therrien et al, 2021). The performance of the hybrid assembly approaches to reconstruct high quality pathogen genomes warrants further improvements in both the sequencing platforms and the bioinformatic pipelines.…”

“…An open‐source software tool, Unicycler, has been developed for assembling bacterial genomes from a combination of short reads and long reads (Chen, Erickson et al., 2020; Wick et al., 2017). While hybrid assembly's approaches were found to generate fragmentation and accuracy biases based upon the initial assembly strategy, they delivered promising results in terms of genome coverage for several pathogenic bacteria relevant to the food industry (Chen, Kuang et al., 2020; Commichaux et al., 2021; Neal‐McKinney et al., 2021; Therrien et al., 2021). The performance of the hybrid assembly approaches to reconstruct high quality pathogen genomes warrants further improvements in both the sequencing platforms and the bioinformatic pipelines.…”