4 STEC Strains from Swine
Swine strains were cultured overnight at 37C with shaking at 220 rpm in lysogeny broth (LB) (Thermo Fisher Scientific, Asheville, NC, USA). To maximize total genomic DNA (gDNA) yields, bacterial overnight cultures were diluted to OD600 of 0.03 in fresh LB medium and grown at 37C with shaking at 220 rpm to mid-log phase (OD600~0.5). Total gDNA was extracted using the Monarch HMW DNA Extraction Kit (New England Biolabs, Ipswich, MA, USA). Genomes were sequenced to closure using Nanopore long-read technology (Oxford Nanopore, UK). Sequencing libraries were prepared with Rapid Barcoding Kit (RBK-114) according to the manufacturer's instructions and sequenced on the PromethION platform on R10.4.1 flow cell (FLO-MIN114). Reads in the fastq format were imported into Galaxy v.22.05. Default parameters were used for all software unless specified otherwise. Fastq reads were QC-ed using FastQC (v.0.74+Galaxy0) (http://www.bioinformatics.babraham.ac.uk/projects/fastqc) and assembled with Flye (v.2.9.3). The chromosomal dnaA and plasmid repA genes were designated as the zero point of the closed molecules prior to annotation using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP).