AbstractThe aim of this study was to compare Illumina and Oxford Nanopore Technology (ONT) sequencing data to quantify genetic variation to assess within-outbreak strain relatedness and characterise microevolutionary events in the accessory genomes of a cluster of 23 genetically and epidemiologically linked isolates related to an outbreak of Shiga toxin-producing Escherichia coli O157:H7 caused by the consumption of raw drinking milk. There were seven discrepant variants called between the two technologies, five were false-negative or false-positive variants in the Illumina data and two were false-negative calls in ONT data. After masking horizontally acquired sequences such as prophages, analysis of both short and long-read sequences revealed the 20 isolates linked to the outbreak in 2017 had a maximum SNP distance of one SNP between each other, and a maximum of five SNPs when including three additional strains identified in 2019. Analysis of the ONT data revealed a 47 kbp deletion event in a terminal compound prophage within one sample relative to the remaining samples, and a 0.65 Mbp large chromosomal rearrangement (inversion), within one sample relative to the remaining samples. Furthermore, we detected two bacteriophages encoding the highly pathogenic Shiga toxin (Stx) subtype, Stx2a. One was typical of Stx2a-phage in this sub-lineage (Ic), the other was atypical and inserted into a site usually occupied by Stx2c-encoding phage. Finally, we observed an increase in the size of the pO157 IncFIB plasmid (1.6 kbp) in isolates from 2019 compared to those from 2017, due to the duplication of insertion elements within the plasmids from the more recently isolated strains. The ability to characterize the accessory genome in this way is the first step to understanding the significance of these microevolutionary events and their impact on the genome plasticity and virulence between strains of this zoonotic, foodborne pathogen.