2.3 Whole-genome sequencing and bioinformatics analysis
Based on drug susceptibility testing, a MDR Klebsiella pneumoniaeisolate (named as M297-1) from Red Kangaroo was identified multidrug
resistant (MDR) bacteria, and subjected to WGS using Oxford Nanopore
Technologies (ONT) MinION platform (Biomarker Technologies, China)
(Ashton et al., 2015; Loman, Quick, & Simpson, 2015). The sequencing
was carried out according to the standard protocol provided by ONT, and
the high-quality genomic DNA, was extracted by Nanodrop, Qubit and
0.35% agarose gel electrophoresis for purity, concentration and
integrity. Large fragments of DNA were recovered by BluePippin automatic
nucleic acid recovery system. The library was constructed by ligation
sequencing kit (SQK-LSK109 Ligation Sequencing Kit, Oxford Nanopore
Technologies, UK), and the DNA damage repair and terminal repair,
magnetic bead purification were used to connect, purify again, and the
Qubit library was quantified and sequenced on the machine.
The phylogenetic tree was constructed based on 16s rRNAsKlebsiella pneumoniae isolate multilocus sequence typing (MLST)
was conducted by using MLST 1.8 or PubMLST (http://pubmlst.org/)Plasmid
replicon typing, plasmid multilocus sequence typing ,and identification
of resistance genes were performed using Plasmid Finder
(https://cge.cbs.dtu.dk/services/PlasmidFinder/)
or pMLST 2.0, and Resfinder 2.0 or CARD, respectively. The comparison of
the similarity between plasmids and known plasmids was conducted using
PLSDB databases
(https://ccb-microbe.cs.uni-saarland.de/plsdb/).
Plasmid sequence was annotated with MiGAP
(https://www.migap.org/), and the
genomic structure was compared in EasyFig. The comparative map of
plasmid genome was drawn by the Illustrator for Biological Sequences
(IBS) software v1.0. (Liu et al., 2015) and modified manually.
Transposons and insertion sequences were determined using ISfinder.