PacBio sequencing, annotation and bioinformatic analysis of MGEs genomes

Genomic DNA was isolated and purified from bacterial cultures grown overnight using the Mo Bio Powersoil® DNA Isolation kit (Mo Bio, Carlsbad, CA, USA) or the DNAesy Blood and Tissue kit (Qiagen, Hilden, Germany), according to manufacturer’s instructions. Long-read sequencing was performed on the three E. coli isolates on a PacBio RSII Instrument at the Ramaciotti Centre for Genomics (UNSW, Sydney, Australia). Polishing and assembly of sequenced reads was performed at the Ramaciotti Centre using HGAP and CANU, and plasmids were closed using Circlator⁴⁸. Errors in PacBio assemblies were checked and curated by alignment with Illumina short reads from whole genome sequencing of the E. coli hosts. P1-like genomes were first annotated using RASTtk⁴⁹, then manually curated using BLAST functions⁵⁰, SnapGene (GSL Biotech; available at snapgene.com) and Geneious v.9.1 (https://www.geneious.com). Plasmid virulence and resistance regions were also annotated using web-based software [Center for Genomic Epidemiology, www.genomicepidemiology.org; Galileo^TM AMR (formerly MARA), galileoamr.arcbio.com/mara/⁵¹]. All allelic variants of IS26⁵² detected in pTZ20_1P are referred to as IS26 throughout. Comparisons with the reference P1 genome (P1 mod749::IS5 c1.100 mutant; GenBank NC_005486)¹⁹ were visualized using EasyFig.⁵³.