A phylogenetic method to perform genome-wide association studies in microbes that accounts for population structure and recombination

Genome-Wide Association Studies (GWAS) in microbial organisms have the potential to vastly improve the way we understand, manage, and treat infectious diseases. Yet, microbial GWAS methods established thus far remain insufficiently able to capitalise on the growing wealth of bacterial and viral genetic sequence data. Facing clonal population structure and homologous recombination, existing GWAS methods struggle to achieve both the precision necessary to reject spurious findings and the power required to detect associations in microbes. In this paper, we introduce a novel phylogenetic approach that has been tailor-made for microbial GWAS, which is applicable to organisms ranging from purely clonal to frequently recombining, and to both binary and continuous phenotypes. Our approach is robust to the confounding effects of both population structure and recombination, while maintaining high statistical power to detect associations. Thorough testing via application to simulated data provides strong support for the power and specificity of our approach and demonstrates the advantages offered over alternative cluster-based and dimension-reduction methods. Two applications to Neisseria meningitidis illustrate the versatility and potential of our method, confirming previously-identified penicillin resistance loci and resulting in the identification of both well-characterised and novel drivers of invasive disease. Our method is implemented as an open-source R package called treeWAS which is freely available at https://github.com/caitiecollins/treeWAS.Author summary: Measurable differences often exist within a microbial population, with important ecological or epidemiological consequences. Examples include differences in growth rates, host range, transmissibility, antimicrobial resistance, virulence, etc. Understanding the genetic factors involved in these phenotypic properties is a crucial aim in microbial genomics. A fundamental approach for doing so is to perform a Genome-Wide Association Study (GWAS), where genomes are compared to search for genetic markers systematically correlated with the property of interest. If this strategy were implemented naively in microbes, it could lead to spurious results due to the confounding effects of population structure and recombination. Here we present treeWAS, a new phylogenetic method to perform microbial GWAS that avoids these pitfalls. We show, using simulated datasets, that treeWAS is able to distinguish between genetic markers that are truly associated with the property of interest and those that are not. Furthermore, we demonstrate that treeWAS offers advantages in both sensitivity and specificity over alternative cluster-based and dimension-reduction techniques. We also showcase treeWAS in two applications to real datasets from N. meningitidis. We have developed an easy-to-use implementation of treeWAS in the R environment, which should be useful to a wide range of researchers in microbial genomics.