2.2. Genome sequencing

In this article, 29 K. pneumoniae isolates collected between 09/2014 and 07/2019, mainly at the Department of Internal Medicine, Hematology and Oncology at the University Hospital Brno, were analyzed. The high molecular weight DNA was extracted using the MagAttract HMW DNAKit (Qiagen, Venlo, NL), and the NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA) was employed to measure the purity of the extracted DNA. The DNA concentration was checked by Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Wilmington, DE, United States) and using Agilent 4200 TapeStation (Agilent Technologies, Santa Clara, CA, USA) the proper length of the isolated DNA was checked. The Rapid Barcoding Kit (Oxford Nanopore Technologies, Oxford, UK) was used to prepare the sequencing library for 27 K. pneumoniae isolates. For the remaining two isolates, the Ligation Sequencing 1D Kit (Oxford Nanopore Technologies, Oxford, UK) was used to prepare the library for Oxford Nanopore sequencing. The sequencing was performed using the MinION sequencing platform (Oxford Nanopore Technologies, Oxford, UK) with R9.4.1 flowcells.

The sequenced genomes were basecalled and, in the case of the pooled library, separated according to barcodes using Guppy software (3.4.4+a296acb). The data quality was checked using PycoQC (v2.2.3, Leger and Leonardi, 2019). See Supplementary Table 1 for detailed information about each sequencing run. The analyzed datasets can be found in the National Center for Biotechnology Information Sequence Read Archive database under a BioProject with accession number PRJNA786743.