ProkEvo: an automated, reproducible, and scalable framework for high-throughput bacterial population genomics analyses

Abstract

Whole Genome Sequence (WGS) data from bacterial species is used for a variety of applications ranging from basic microbiological research, diagnostics, and epidemiological surveillance. The availability of WGS data from hundreds of thousands of individual isolates of individual microbial species poses a tremendous opportunity for discovery and hypothesis-generating research into ecology and evolution of these microorganisms. Scalability and user-friendliness of existing pipelines for population-scale inquiry, however, limit applications of systematic, population-scale approaches. Here, we present ProkEvo, an automated, scalable, and open-source framework for bacterial population genomics analyses using WGS data. ProkEvo was specifically developed to achieve the following goals: 1) Automation and scaling of complex combinations of computational analyses for many thousands of bacterial genomes from inputs of raw Illumina paired-end sequence reads; 2) Use of workflow management systems (WMS) such as Pegasus WMS to ensure reproducibility, scalability, modularity, fault-tolerance, and robust file management throughout the process; 3) Use of high-performance and high-throughput computational platforms; 4) Generation of hierarchical population-based genotypes at different scales of resolution based on combinations of multi-locus and Bayesian statistical approaches for classification; 5) Detection of antimicrobial resistance (AMR) genes, putative virulence factors, and plasmids from curated databases and association with genotypic classifications; and 6) Production of pan-genome annotations and data compilation that can be utilized for downstream analysis. The scalability of ProkEvo was measured with two datasets comprising significantly different numbers of input genomes (one with ~2,400 genomes, and the second with ~23,000 genomes). Depending on the dataset and the computational platform used, the running time of ProkEvo varied from ~3-26 days. ProkEvo can be used with virtually any bacterial species and the Pegasus WMS facilitates addition or removal of programs from the workflow or modification of options within them. All the dependencies of ProkEvo can be distributed via conda environment or Docker image. To demonstrate versatility of the ProkEvo platform, we performed population-based analyses from available genomes of three distinct pathogenic bacterial species as individual case studies (three serovars of Salmonella enterica, as well as Campylobacter jejuni and Staphylococcus aureus). The specific case studies used reproducible Python and R scripts documented in Jupyter Notebooks and collectively illustrate how hierarchical analyses of population structures, genotype frequencies, and distribution of specific gene functions can be used to generate novel hypotheses about the evolutionary history and ecological characteristics of specific populations of each pathogen. Collectively, our study shows that ProkEvo presents a viable option for scalable, automated analyses of bacterial populations with powerful applications for basic microbiology research, clinical microbiological diagnostics, and epidemiological surveillance.