Novaseq 6000

A QIAamp PowerFecal Pro DNA Kit (Qiagen, Hilden, Germany) was employed to extract genomic DNA from the KD337-16^T strain. Subsequently, the SQK-LSK109 Ligation Sequencing Kit on a PromethION Flow Cell (R9.4.1) and Illumina NovaSeq 6000 in paired-end (2 × 151 bp) mode was used for Oxford Nanopore Technologies (ONT) sequencing. After the sequences had been decoded and refined, Flye version 2.8.3 was employed for assembly of the valid ONT sequences. The primary contigs were polished with Racon v1.4.22 and the Illumina read alignment results constructed using Minimap2 v2.17. The DDBJ Fast Annotation and Submission Tool was used to annotate the genome [35 (link)]. Methods described elsewhere [36 (link),37 (link),38 (link)] were employed to quantify the digital DNA–DNA hybridization (dDDH), the amino acid identity (AAI), and average nucleotide identity (ANI). The up-to-date bacterial core genes pipeline (http://leb.snu.ac.kr/ubcg2, accessed on 28 October 2022) [39 (link)] and EDGAR platform were utilized to construct phylogenomic trees [40 (link)], whereas the 3ggNOG 4.5 database and carbohydrate-active enzyme (CAZy) database were employed for functional assignment [41 (link),42 (link)]. The OrthoVenn2 webserver was used for pangenome analysis [43 (link)]. Finally, AntiSMASH software (v. 6.0) was employed to predict putative biosynthetic gene clusters [44 (link)].

About 10 g of fresh cabbage leaves were harvested and quick-frozen in liquid nitrogen for DNA isolation using a modified extraction method. Genome sequencing of DNA samples was performed using second-generation Illumina HiSeq and third-generation ONT sequencing technology, after which an Illumina paired-end (PE) library (300–500 bp) and a Nanopore library (8–10 kb) were constructed. Sequencing was performed using NovaSeq 6000 and Nanopore high-throughput sequencing platforms.
The adapters, reads containing over 10% Ns (uncalled bases), duplicated sequences, and low-quality reads (the Phred scores < Q20) were removed. The Phred scores (Q20, Q30) and GC content of the clean data were calculated. SPADES 3.9.0 and Paired-Read Iterative Contig Extension (Price) software were used for genome assembly.
UGENE ORFS Finder was used to annotate the protein-coding genes, and tRNA annotations were submitted to the tRNAscan-se online website for annotation. RNAmmer 1.2 Server (https://services.healthtech.dtu.dk/service.php?RNAmmer-1.2/ (accessed on 2 December 2021)) was used to annotate the rRNAs. After the sequence annotation was finished, it was edited via Sequin to generate a submission file that could be submitted to the GenBank database. The edited GenBank annotation file was submitted to OGDRAW to construct an annotation map.

Genomic DNA was extracted using a genomic DNA purification kit (Generay, Shanghai, China). The concentration and purity of the DNA in the samples were detected using a NanoDrop 2000 spectrophotometer (Thermo Fisher, Waltham, MA, USA) and a Qubit 3.0 fluorometer (Invitrogen, USA), respectively. WGS was performed using the Illumina NovaSeq 6000 and the Nanopore sequencing GridION platforms. Sequence assembly was then performed with Unicycler v. 0.4.3. In addition, the NCBI prokaryotic genome annotation pipeline was used for gene annotation. Acquired AMR genes were predicted using ResFinder 4.1 (https://cge.cbs.dtu.dk/services/ResFinder/). The MLST information of the isolates was extracted from assembled genomes. The plasmid sequence was annotated using RAST (http://rast.nmpdr.org/) followed by manual review using BLAST and the plasmid replicon genotype was identified using Plasmid Finder (https://cge.cbs.dtu.dk/services/PlasmidFinder/) (35 (link)). Insertion sequence (IS) elements were identified using ISfinder (https://isfinder.biotoul.fr/) (36 (link)). The transposon name was assigned by the Tn Registry website curators (https://transposon.lstmed.ac.uk/). Comparative analysis and plasmid maps were generated by BRIG (37 (link)).