Sequencing and Assembly of Bacterial Genomes

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.