100 Chapter 6 Whole-Genome Bioinformatics Analysis of Short-Read Sequencing Data The presence of acquired resistance genes was identified by uploading assembled genomes to the ResFinder web service of the Center for Genomic Epidemiology (version 3.1) (Zankari et al. 2012). The presence of plasmid replicons and the typing of a specific IncI plasmid was performed using pMLST (version 2.0) (Alessandra Carattoli et al. 2014). The genomes were selected based on a 100% match to blaCMY-2 and IncI-pST12. Typing of a specific multi locus sequence type (MLST) was performed using the MLST web service of the Center for Genomic Epidemiology (version 2.0), and fim typing was performed using FimTyper (version 1.0), Center for Genomic Epidemiology (Camacho et al. 2009; Larsen et al. 2012). Long-Read Sequencing and Hybrid Assembly No more than two isolates of the same flock or patient belonging to the same MLST were selected for further long-read sequencing. All isolates were long-read sequenced on a MinION sequencer using the FLOMIN106D flow cell and the Rapid Barcoding Sequencing Kit SQK RBK004 according to the standard protocol provided by the manufacturer (Oxford Nanopore Technologies, Oxford, UK). A hybrid assembly of long-read and short-read sequence data was performed using Unicycler v.0.8.4 (Wick et al. 2017). Whole-genome MLST (wgMLST) (core and accessory genome) was performed for all isolates using Ridom SeqSphere+, version 4.1.9 (Ridom, Münster, Germany). Species-specific wgMLST typing schemes were used as described previously (Marjolein F Q Kluytmans-Van Den Bergh et al. 2016). The pairwise genetic difference between isolates of the same species was calculated by dividing the total number of allele differences by the total number of shared alleles from the typing scheme present in both sequences, using a pairwise ignoring missing values approach. Genetic relatedness was determined using the thresholds for wgMLST-based genetic distance of 0.0095, as described previously (Marjolein F Q Kluytmans-Van Den Bergh et al. 2016). Plasmid Analysis The genomes created using the hybrid assembly were uploaded to the online bioinformatics tools ResFinder v.2.1, VirulenceFinder v.1.2 and PlasmidFinder v.1.2. (Center for Genomic Epidemiology, Technical University of Denmark, Lingby, Denmark) (Zankari et al. 2012; Alessandra Carattoli et al. 2014; Joensen et al. 2014). Circular components created by the hybrid assembly that were smaller than 1000 kb and that contained an IncI1-pST12 plasmid replicon and a blaCMY-2 gene were extracted from
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