149 In silico estimates of plasmid copy number and increased resistance in CMY-2-producing E. coli Kluytmans-Van Den Bergh et al. 2016). De novo assembly was performed using CLC Genomics Workbench 8.5.1 (Qiagen), with optimal word sizes based on the maximum N50 (the largest scaffold length, N, such that 50% of the assembled genome size is contained in scaffolds with a length of at least N) value. Assembled genomes were excluded from further analyses if they did not meet the previously described quality criteria for coverage (mean matched read depth), the number of scaffolds, N50, maximum scaffold length, and percentage of expected genome size (Marjolein F Q Kluytmans-Van Den Bergh et al. 2016). Analysis of assembled genomes Assembled genomes were uploaded to the online bioinformatics tools ResFinder v2.1 and MLST v1.8 (Center for Genomic Epidemiology, Technical University of Denmark, Lyngby, Denmark) (Zankari et al. 2012; Larsen et al. 2012). Acquired resistance genes were reported when at least 60% of the length of the best matching gene in the ResFinder database was covered with a sequence identity of at least 90%. Conventional MLST sequence types were based on the Achtman MLST scheme 23. For each assembled genome, the promoter/attenuator region of the chromosomal AmpC (cAmpC) gene was aligned with that of E. coli K-12 MG1655 (GenBank accession number U00096) using CLC Genomics Workbench 8.5.1. wgMLST was performed using Ridom SeqSphere+ (Ridom, Münster, Germany) as described previously (Marjolein F Q Kluytmans-Van Den Bergh et al. 2016). CLC Genomics Workbench 8.5.1. (Qiagen) was used to obtain the coverage for 1) the whole genome assembly; 2) the scaffolds containing the blaCMY-2 gene, and 3) the scaffolds containing either one of the seven E. coli MLST genes (adk, fumC, gyrB, icd, mdh, purA and recA) (Wirth et al. 2006). The ratio between the coverage of the blaCMY2-encoding scaffold and the coverage of the whole genome assembly was used as an in-silico estimate for the plasmid copy number (ePCN). The ratio between the coverage of the MLST-gene-containing scaffolds and the coverage of the whole genome was used to validate the use of the coverage of the whole genome assembly as a reference in the calculation of the ePCN. Statistical analysis The Mann-Whitney U test was used to test for differences in median ePCN value between ‘low-MIC isolates’ and ‘high-MIC isolates’. Data was analysed with Statistical Package for Social Science software (SPSS; IBM Corp., Armonk, New York, US; version 22). 8
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