Summary Chlamydia trachomatis infections in humans cause diseases of the eye and genital tract that affect millions of people annually. Our understanding of C. trachomatis biology is complicated by its obligate intracellular nature and a developing, but limited genetic tool kit. Because of the high level of conservation among C. trachomatis strains found in patients, next-gen sequencing has evolved into a straightforward tool for analysis of genetic variability of the pathogen. Our laboratory has a goal of monitoring genomic changes in C. trachomatis isolates circulating in patient populations. This monitoring is significantly affected by the lack of recent cultured strains, which are becoming increasingly rare as clinics move completely to nucleic acid amplification testing for chlamydial diagnosis. To address our goal in the current diagnostics climate, we propose to develop a robotics-based approach for evaluating contemporary strain variability, with deidentified remnant clinical samples as a source of material. Bioinformatic analysis of all published C. trachomatis genomes has identified a large library of candidate oligonucleotide primers that can be used to generate material for sequencing all variable regions of the genome, and we have demonstrated that remnant diagnostic material is suitable for a PCR-based approach. Draft analyses indicate that all indels and over half of all nonconservative base changes can be assessed in a set of 96 individual PCR reactions. In Aim 1, we will carefully evaluate different primer pairs to identify a collection that will cover a maximum fraction of the genome in a single tray-based amplification format. In Aim 2, we will test our identified primer set in three increasingly complicated robotics-based assays to assess the utility of the system for examining global genetic variation in a single high-throughput experiment. The final product of this project will be a flexible process that can amplify and sequence large collections of specimens and will take approximately one week to complete. We anticipate that this platform will be useful to basic researchers and clinician scientists exploring the connections between chlamydial genotype and clinical presentation, and will serve as a model for employing the approach with other challenging microorganisms for which diagnosis of infection does not include a culture step.