Single-molecule nanopore sequencing reveals extreme target copy number heterogeneity in arylomycin-resistant mutants
Hany S. Girgis, Cory D. DuPai, Jessica Lund, Janina Reeder, Joseph Guillory, Steffen Durinck, Yuxin Liang, Joshua S. Kaminker, Peter A. Smith, Elizabeth Skippington
Abstract
Significance Genetic heterogeneity is a significant driver of antibiotic resistance in bacteria. Understanding copy number (CN) heterogeneity is important because minority subclones with increased CN can drive resistance during antibiotic exposure, but revert and escape detection during clinical susceptibility testing. Despite its clinical relevance, CN variation has eluded quantification at single-molecule resolution. Here, we report nanopore sequencing of arylomycin-resistant mutants carrying tandem repeats ranging in size from 4.8 to 50.0 kb and encompassing the arylomycin target gene lepB . Reads spanning individual repeat arrays show vast differences in CN, underscoring the importance of amplifications in driving the emergence of genetic heterogeneity. This is a direct observation of cell-to-cell CN differences in an antibiotic-resistant bacterial population.