C. kerstersii 8943 was cultivated in tryptic soy broth, which contains 17.0 g/L tryptone (pancreatic digest of casein), 3.0 g/L soytone (peptic digest of soybean), 2.5 g/L glucose, 5.0 g/L sodium chloride and 2.5 g/L dipotassium phosphate. After shaking at 37 °C for 24 h, bacterial cells were harvested by centrifugation at 5,000 rpm for 10 min. Genomic DNA was extracted using QIAamp DNA Mini Kit (Qiagen, Germany) according to manufacturer’s instructions. The quality and integrity of genomic DNA was assessed using 1% agarose gel electrophoresis and densitometry compared to the appropriate size standards. Meanwhile, DNA yield and purity were measured using NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific, USA) and Qubit®2.0 fluorometer (Thermo Fisher Scientific, USA).

Qualified genomic DNA was sheared using a Covaris® g-TUBE® shearing device (Covaris, USA) (>10 kb insert sizes). After shearing, the approximate sizes of the DNA were determined using Agilent® 2100 Bioanalyzer (Agilent Technologies, USA). The fragmented DNA was then purified using 0.45×AMPure® PB beads (Pacific Biosciences, USA). The ends of the fragmented DNA were repaired using the PacBio Template Prep Kit (Pacific Biosciences, USA) before ligating to the hairpins (SMRTbell™ templates). The resulting SMRTbell library was quantitated via Qubit. Before sequencing, sequencing primers were annealed to both ends of the SMRTbell template and DNA sequencing polymerases were bound to the templates to form the template-polymerase complex. Single-molecule real-time (SMRT®) sequencing was performed using a Pacific Biosciences RSII sequencer (PacBio, Menlo Park, CA) according to the manufacturer’s instructuions (MagBead Standard Seq v2 loading, 1×180 min movie) using P4-C2 chemistry.

Hierarchical Genome Assembly Process (HGAP) pipeline was used to a generate high quality de novo assembly of the genome with default parameters⁷. Shorter reads were aligned against the longest reads to correct random errors and generate the pre-assembled reads that were both long and of high accuracy. The quality of the assembled genome was assessed using CheckM v1.0.9⁸. The circulation of the assembled genome was verified by aligning the complete genome with the draft genome of C. kerstersii 8943 (ASM129444v1). Open reading frames (ORFs) were predicted using Glimmer v3.02. rRNAs and tRNAs were predicted using RNAmmer⁹ and tRNAscan-SE¹⁰, respectively. The phylogenetic tree was constructed according to the neighbor-joining method using Molecular Evolutionary Genetics Analysis (MEGA) 7.0 software¹¹. Antibiotic resistance genes were annotated using BLAST-2.7.1+ program¹² against the Antibiotic Resistance Gene-ANNOTation (ARG-ANNOT) database¹³ with an e-value cut-off of 1e-5 and identity cut-off of 90%. Prophage regions were identified by PHAge Search Tool Enhanced Release (PHASTER)¹⁴. The circular map of the genome was generated using DNAPlotter software¹⁵ and multiple genome alignment was performed using BLAST Ring Image Generator (BRIG)¹⁶. The genomic average nucleotide identity (ANI) was calculated using Orthologous Average Nucleotide Identity Tool (OAT)¹⁷.