Whole-Genome Sequencing of E. coli

Total gDNA was extracted from an overnight culture (2 ml) on a QIAcube automated system (Qiagen). Following extraction, gDNA was quantified by fluorometric methods using a Qubit (ThermoFisher Scientific), with quality ratios of gDNA (A260/280 and 260/230) determined via Nanodrop (ThermoFisher Scientific). Genomic DNA libraries are prepared for whole-genome sequencing using the NexteraXT kit (Illumina), as described by the manufacturer. Paired end sequencing was performed using the Illumina MiSeq platform (MiSeq Reagent V3 Kit; 2 × 300 cycles). For each E. coli isolate, at least 80× coverage was generated. Raw sequence reads were trimmed using Trim Galore and the genomes were de novo-assembled into contigs using SPAdes (3.9.0) with pre-defined kmers set. Raw reads were also assembled with Geneious (10.0.9; Biomatters Ltd.) de novo assembler, set at medium sensitivity for analysis of paired Illumina reads. Geneious was used to map both sets of contigs to reference genes identified by closest BLAST homology and was also used to annotate genes from closest homologues in NCBI Genome database. Resistance genes were identified using Resfinder within CGE^{59 (link)}, and wgMLST profiles were generated using the CGE platform coupled with the PubMLST.org database^{60 (link)}. Plasmids were identified within the genome assembly and typed using Plasmidfinder^{61 (link)}.