Dneasy ultraclean 96 kit

Whole genome sequencing and sequence analysis was done as previously described [19 (link)]. Briefly, DNA was extracted from bacterial cultures using a DNeasy UltraClean 96 Kit (Qiagen, Venlo, Netherlands). Library preparation and sequencing was performed by a commercial service provider (Novogene, Cambridge, United Kingdom) using Illumina chemistry. Sequencing was carried out on a NovaSeq 6000 flow cell using a paired-end sequencing strategy of 2 × 150 bp. Analysis of phylogenetic relatedness was carried out using the pan-genome pipeline Roary [20 (link)]. In silico screening for antimicrobial resistance genes was performed with ABRicate (accessible at https://github.com/tseemann/abricate) using the Comprehensive Antibiotic Resistance Database (CARD) [21 (link)] as reference. We also used ABRicate to screen the genomes of K. pneumoniae isolates for hypervirulence genes by searching against gene entries of hypervirulence plasmid pLVPK (NC_005249.1) as the reference. Plasmid-related sequences were analysed with RFplasmid (accessible at http://klif.uu.nl/rfplasmid) and plasmidMLST (accessible at https://pubmlst.org/bacteria/plasmid-mlst). All sequence data generated for this study were deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra) and are available under BioProject accession number PRJNA891092.

DNA of cultured bacteria was extracted using DNeasy UltraClean 96 kit (Qiagen, Venlo, Netherlands). Library preparation and isolates sequencing in this study were either carried out at the Twincore (Center of Clinical and Experimental Infection Research, Hanover, Germany) or were performed by a commercial service provider (Novogene, Cambridge, UK). All isolates were sequenced using Illumina chemistry utilizing a paired-end sequencing strategy of either 2 × 150 bp or 2 × 250 bp.
Postsequencing, a quality assessment of all FastQ files was performed using FastQC version 0.11.8 (40 ). Remaining partial adapter sequences were removed with Cutadapt (41 (link)). De novo assembly was carried out using Unicycler version 0.4.8-beta as an optimizer for SPAdes (42 (link)). Assembly statistics were assessed using R (43 ) version 3.4.4 with the package SeqinR (44 (link)). As quality criteria, (i) total assembled length of each bacterial isolate had to fall between 6 and 7.5 Mbp, (ii) N₉₀ values of assemblies had to be above 10 kb, and (iii) the total amount of contigs of an assembly larger than 1 kb had to be below 300.