Project Summary Sensitive Treponema pallidum genome recovery through tiling amplicon sequencing Genomic epidemiology is now a standard part of the public health response to outbreaks of bacterial pathogens. For most bacteria, genome recovery is performed via shotgun sequencing of clinical isolates. Genome recovery for Treponema pallidum, the causative agent of syphilis, is more complicated due to the lack of routine culture for the organism in clinical labs. Expensive reagents and complicated protocols are required to perform hybridization capture, which is not standard in clinical or public health microbiology laboratories. Even then, the analytical sensitivity of hybridization capture does not meet the low levels of T. pallidum DNA present in most clinical samples, leading to failure of complete genome recovery in over half of clinical specimens that test PCR positive for T. pallidum DNA. Here, we propose to optimize methods to recover T. pallidum genomes from diverse specimens to allow for greater analytical sensitivity and scale. Specifically, based on recent data from hundreds of newly sequenced T. pallidum genomes, we will design a tiling amplicon sequencing approach for T. pallidum genome recovery, using 2- 3kb sized PCR amplicons compatible with both long-read and short-read sequencing. We will then fully validate this tiling amplicon library generation approach using both cultured isolates and diverse clinical T. pallidum specimens taken from across the world to determine its analytical sensitivity, specificity, precision, and accuracy. Finally, we will widely share our standard operation procedures and reagents for executing this work in research and public health laboratories across the world. The proposed work will extend the analytical sensitivity, ease of workflow, and cost-effectiveness for T. pallidum genome recovery, enabling genomic epidemiology to inform outbreak response and prevention for syphilis.