123 Genome-wide analysis in E. coli unravels homoplasy associated with cefotaxime resistance g-TUBEs for 30 s at 11000 r.p.m. Each DNA sample was separated into two aliquots. Size selection was performed using a 0.75% agarose cassette and marker S1 on the BluePippin system (Sage Science) to obtain either 4–8 kb or 4–12 kb DNA fragments. This size selection was chosen to maintain all DNA fragments, including these originating from plasmids (data not used in this study). Library preparation was performed using the PacBio SMRTbell template prep kit 1.0 (Pacific Biosciences). For cost-effectiveness, samples were barcoded and pooled with other samples that are not relevant for this study. Sequencing was conducted using the PacBio Sequel I (Pacific Biosciences) on a Sequel SMRT Cell 1M v2 (Pacific Biosciences) with a movie time of 10 h (and 186 min preextension time). Subreads per sample were obtained by extracting the bam files using SMRT Link version 5.1.0.26412 (Pacific Biosciences). Chromosomal reconstruction using de novo hybrid assembly To obtain a full-length chromosome, Unicycler version 0.4.7 (settings: --mode bold) was used, combining Illumina NextSeq 500 2×150 bp paired-end reads with PacBio Sequel SMRT subreads (Wick et al. 2017). Because Unicycler requires fasta reads as input, the subreads in bam format were converted to fasta by using bam2fasta version 1.1.1 from pbbioconda (https://github.com/PacificBiosciences/pbbioconda) prior to de novo hybrid assembly. The full circular chromosome was uploaded to the National Center for Biotechnology Information (NCBI) database and annotated using NCBI Prokaryotic Genome Annotation Pipeline (PGAP) version 4.10 (Tatusova et al. 2016; Haft et al. 2018). SNP analysis using E. coli reference ampC_0069 Alignment of Illumina reads and SNP calling was performed for all isolates to the reference chromosome of E. coli isolate ampC_0069 using Snippy version 4.3.6 (https:// github.com/tseemann/snippy). A full-length alignment (fullSNP) and a coreSNP alignment containing SNP positions shared among all isolates were generated by using snippy-core version 4.3.6 (https://github.com/tseemann/snippy). Inferring of phylogeny A phylogenetic tree was inferred by using the coreSNP alignment as input for FastTree(MP) version 2.1.3 SSE3 (settings: -nt -gtr) (M. N. Price, Dehal, and Arkin 2010). Detection of homoplasy The consistency index for all nucleotide positions on the chromosome was calculated using HomoplasyFinder version 0.0.0.9000 (Crispell, Balaz, and Gordon 2019). The 7
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