DNA obtained from the ancestral strains and evolved lines that showed PFGE variation were end-repaired (NEBnext ultra II end repair kit, New England Biolabs, MA, USA) and cleaned up with 1× AmPure beads (BeckmannCoulter, USA). Native barcode ligation (BC02-05) was performed with NEB blunt/TA ligase (New England Biolabs, MA, USA) and cleaned with 0.5× AmPure beads. Qubit quantified barcode ligated DNA samples were pooled at equi-molar concentration to attain 1 µg pooled sample. Adapter ligation (BAM) was performed for 15 min using NEBNext Quick Ligation Module (New England Biolabs, MA, USA). Library mix was cleaned up using 0.4X AmPure beads (Beckmann Coulter, USA) and library was eluted in 15 µl of elution buffer and used for sequencing. Sequencing was performed on MinION Mk1b (Oxford Nanopore Technologies, Oxford, GBR) using SpotON flow cell (FLO-MIN107) in a 48 h sequencing protocol on MinKNOW 1.7.7. Base calling was performed using Albacore v.1.2.6. Reads were processed by albacore and poretools (Loman and Quinlan 2014) (link) for converting fast5 files into fasta format.