Genome sequencing and assembly

Genomic DNA of the strains sequenced in this study was extracted and purified using Genomic-tips 100/G and a genomic DNA buffer set (Qiagen) from overnight cultures in lysogeny broth (LB) at 37 °C following the manufacturer’s protocol, with minor modifications including the addition of SDS (final concentration 1 %) after adding buffer B2 and incubation for 1 h at 50 °C. For short-read sequencing, libraries were prepared using the Nextera XT DNA sample prep kit (Illumina) or NEBNext Ultra II FS DNA library preparation kit (New England Biolabs) and sequenced using the Illumina MiSeq platform to generate paired-end sequence reads (301 bp ×2). Reads were trimmed by Platanus trim [27] with default parameters and assembled by Platanus_B_v1.3.1 [28]. Scaffolds ≥300 bp were used in this study.

To determine complete genome sequences, size selection of purified DNA was performed using magnetic beads (AMPure XP; Beckman Coulter) to obtain longer DNA fragments. Sequencing libraries were prepared using a rapid barcoding kit (SQK-RBK004), sequenced using the R9.4.1 flow cell with the Oxford Nanopore Technologies (ONT) MinION platform, and base-called using Guppy GPU ver. 3.4.5. (ONT). Reads were trimmed using NanoFilt [29] with the following parameters: minimum length=7 000 bp, minimum quality score=10 and 5'-terminal 100 bases cutting. For sequencing of three strains (93_161312, {"type":"entrez-protein","attrs":{"text":"CEC13091","term_id":"815592253","term_text":"CEC13091"}}CEC13091 and F690), only≥15 kb reads were used for assembly to gain better results. For sequencing of strain F765, the minimum length was changed to 2000 bp to salvage its small plasmid sequence. ONT read assembly and polishing were performed using the microPIPE pipeline [30]. In brief, trimmed ONT reads were assembled using Flye (v2.8.3) [31] with the option ‘--plasmids’ and polished with ONT reads using four iterations of Racon (v1.4.20) [32] followed by one iteration of Medaka (v1.4.3) (GitHub – https://github.com/nanoporetech/medaka). Output contigs were further refined with Illumina short reads using NextPolish (v1.3.1) (GitHub – https://github.com/Nextomics/NextPolish). As the chromosome of strain 93_161312 and the plasmids of strain F690 were not circularized, manual curation was performed using Minimap2 [33], Integrative Genomics Viewer (igv) [34] and GenomeMatcher v3.0.2 [35] to obtain their circular chromosome and plasmid sequences.