Minion system

Circular low-molecular-weight DNA was rolling circle amplified with the TempliPhi amplification kit (Cytiva). Amplified DNA was digested with BamHI to release linear single genome-length molecules. The digested DNA was separated on a 1% agarose gel, and the ∼5-kb genomic DNA band was harvested with a QIAquick gel extraction kit (Qiagen). The DNA library was prepared according to the manual for the ligation sequencing kit (Nanopore). Long-read DNA sequences were read with the Nanopore MinION system in our laboratory.

The genomic DNA of the K. pneumoniae NB4 and NB5 strains was obtained using a QIAamp DNA MiniKit (Qiagen, Valencia, CA, USA) following the manufacturer’s recommendations. The combination Oxford Nanopore (MinION system, Nanopore, Oxford, UK) and Illumina sequencing (NovaSeq system, Illumina Inc, San Diego, U.S.A) were used to achieve the complete chromosomes and plasmid sequences, respectively.
The Illumina reads and Nanopore reads were assembled using the hybird assembly tool Unicycler version 0.4.8 (Wick et al., 2017 (link)). Annotation of the plasmid genomes was performed using the RAST annotation website server (http://rast.nmpdr.org/rast/cgi).
Antibiotic resistance genes (ARGs), plasmid replicon types, and sequence type of the strains were obtained by the ResFinder 4.1, PlasmidFinder 1.3 and MLST 2.1 servers, which are available at the Center for Genomic Epidemiology (http://www.genomicepidemiology.org/). The virulence factors were identified using the kleborate software (https://github.com/katholt/Kleborate). BRIG and Easyfig were used to visualize the plasmid comparisons and genetic context comparisons, respectively (Alikhan et al., 2011 (link); Sullivan et al., 2011 (link)).