Rapid barcoding kit

DNA extraction was performed for each of the isolated samples using the GenElute™ Bacterial Genomic DNA kit (Sigma-Aldrich). After gDNA extraction, samples were pooled to obtain samples of 400 ng gDNA to identify conserved driver mutations. Library preparation and barcoding was completed using the Rapid Barcoding Kit (Nanopore). A Nanopore MinION Sequencer was used to complete bacterial whole genome sequencing using default settings. After sequencing, quality control was performed using Epi2ME (Nanopore). Reads were trimmed using Porechop²⁹ and aligned to a reference P. aeruginosa PAO1 genome (GCF_000006765.1^{30 (link)}) using graphmap^{31 (link)}. Reads were sorted and indexed using samtools^{32 (link)}. Structural variants were identified using sniffles^{33 (link)} and SNPs were identified using BCFtools^{34 (link)}. SNPs were selected by a quality score > 20.

Transconjugants were sequenced by a combination of Oxford Nanopore and Illumina MiSeq technologies. Complete genomic DNA (gDNA) was purified using the Qiagen Genomic-Tip 100 kit according to the manufacturer’s directions. Library preparations for Nanopore sequencing were performed with the Rapid barcoding kit (Nanopore) according to the manufacturer’s recommendations. Multiplexed libraries were concentrated using AMPure XP beads (Nanopore) and sequenced in a flow cell until no active pores remained. MiSeq libraries were prepared and sequenced by 300-bp paired-end read lengths using a Nextera XT DNA library preparation kit (Illumina) according to the manufacturer’s instructions. De novo assembly of the Nanopore data was performed using the long-read support function in CLC Genomics Workbench v.20 (Qiagen). Contigs were reanalyzed by reference assembly using MiSeq data with CLC Genomics Workbench v.20 including the Microbial Analysis Pro suite (Qiagen). Detection of resistance genes and plasmid replicons was done by submitting de novo-assembled contigs to the online resources ResFinder (66 (link)) and PlasmidFinder (67 (link)), respectively, at the Center for Genomic Epidemiology, DTU, Denmark.

PacBio RSII sequencing was performed by Macrogen Oceania, South Korea, using gDNA extracted with the CTAB protocol. For Oxford Nanopore sequencing, libraries were prepared from gDNA extracted with the DNeasy Blood & Tissue Kit using the Nanopore Rapid Barcoding Kit according to the manufacturer’s instructions. Nanopore data were obtained using a MinION R9.4.1 flow cell. Base-calling was performed using Guppy (Oxford Nanopore Technologies) and demultiplexing using DeepBinner (47 (link)). Tombo was used to detect modified bases by comparison to the reference signal models for unmodified, 6mA or 5mC bases (48 (link)).