Genome sequencing and bioinformatics analysis

Total genomic DNA from microbial isolates was sequenced using a Roche GS 454 FLX system and standard LR 70 chemistries. Illumina Nextera XT library preparation and sequencing (on a MiSeq with V3 chemistry and 300 bp paired end reads) services were provided by the Microbial Genome Sequencing Center (SeqCenter, Pittsburgh, PA, United States). Adaptors and low-quality bases were trimmed from raw reads with trimmomatic v 0.36. Long read sequencing was performed using the Oxford Nanopore platform. These sequence reads were used in combination with Illumina reads to improve accuracy at the nucleotide level, assemble plasmids, and for closing genomes. De novo assembly was performed using SPAdes v 3.13.0 (Bankevich et al., 2012). Plasmid sequences were identified using plasmidSPAdes and classified using the DoriC 10.0 and OriFinder, databases of replication origins in prokaryotic genomes (Antipov et al., 2016; Luo et al., 2019; Luo and Gao, 2019). The location of the oriT in the plasmids, if present, was predicted using oriT Finder with Blast E-value cut-off set to 0.01 (Li et al., 2018).

Initial genome and plasmid annotation was performed with Prokka v 1.12 (Seemann, 2014) and manually checked using the genome viewer Artemis (Carver et al., 2012) and Geneious (v11.1.5, URL: http://www.geneious.com) together with blastp. Blastn and tblastx were used for plasmid comparison, using both NCBI tools and within BLAST Ring Image Generator, BRIG v0.95, (URL: http://brig.sourceforge.net/). (Alikhan et al., 2011).