A method to correct for local alterations in DNA copy number that bias functional genomics assays applied to antibiotic-treated bacteria

Abstract

Functional genomics techniques, such as transposon insertion sequencing and RNA sequencing, are key to studying relative differences in mutant fitness or gene expression under selective conditions. However, certain stress conditions, mutations, or antibiotics can directly interfere with DNA synthesis, resulting in systematic changes in local DNA copy number along the chromosome. This can lead to artefacts in sequencing-based functional genomics data when comparing antibiotic treatment to an unstressed control, with relative differences in gene-wise read counts being the result of alterations in chromosomal replication dynamics rather than selection or direct gene regulation. We term this artefact chromosomal location bias and implement a principled statistical approach to correct for it by calculating local normalization factors along the chromosome. These normalization factors are then directly incorporated in statistical analyses using standard RNA-sequencing analysis methods without modifying the read counts themselves, preserving important information about the mean-variance relationship in the data. We illustrate the utility of this approach by generating and analysing a ciprofloxacin-treated transposon insertion sequencing dataset in Escherichia coli as a case study. We show that ciprofloxacin treatment generates chromosomal location bias in the resulting data, and we further demonstrate that failing to correct for this bias leads to false predictions of mutant drug sensitivity as measured by minimum inhibitory concentrations. We have developed an R package and user-friendly graphical Shiny application, ChromoCorrect, that detects and corrects for chromosomal bias in read count data, enabling the application of functional genomics technologies to the study of antibiotic stress. IMPORTANCEAltered gene dosage due to changes in DNA replication has been observed under a variety of stresses with a variety of experimental techniques. However, the implications of changes in gene dosage for sequencing-based functional genomics assays are rarely considered. We present a statistically principled approach to correcting for the effect of changes in gene dosage, enabling testing for differences in the fitness effects or regulation of individual genes in the presence of confounding differences in DNA copy number. We show that failing to correct for these effects can lead to incorrect predictions of resistance phenotype when applying functional genomics assays to investigate antibiotic stress, and we provide a user-friendly application to detect and correct for changes in DNA copy number.