125 Genome-wide analysis in E. coli unravels homoplasy associated with cefotaxime resistance Visualization of data The interactive tree of life web-based tool (iTOL) version 5.3 was used to visualize the phylogenetic tree (Letunic and Bork 2019). Information about CTX resistance, presence of the pampC gene, campC hyperproduction as defined, MLST and phylogroup, as well as alignments of promoter and attenuator region, were incorporated into the visualization. The sequence logo of the promoter and the attenuator alignment were generated using the web-based application WebLogo, version 3.7 (http://weblogo.threeplusone.com) (Crooks et al. 2004). A chromosome ideogram of the E. coli isolate ampC_0069 reference chromosome was visualized using the circos software package, version 0.69-8 (Krzywinski et al. 2009). Consistency index scores and significant mutations associated with CTX resistance were plotted in the ideogram. Gubbins results were displayed by using Phandango (Hadfield et al. 2018). Overview of the method A workflow graph of the method is visualized in Fig. 1, using the web-based application yEd Live version 4.4.2 (https://www.yworks.com/yed-live/). Figure 1. Schematic of the workflow used to perform the homoplasy-based association analysis. Starting from the top, (a) the de novo assembly of the NextSeq/MiSeq reads and (b) the hybrid assembly of the reference chromosome ampC_069. On the left side, (c) the alignment of promoter/ attenuator region. In the middle, (d) the coreSNP analysis for the phylogeny used in (e) the homoplasy analysis combined with (f) the fullSNP data, on the right, which was also used for (g) the statistics (Fisher›s exact test and FDR) to relate CTX resistance to SNP positions. (h) Inferring recombination events using Gubbins. 7
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