| Resumo: | Scrub typhus is a febrile disease caused by <em>Orientia tsutsugamushi</em>, transmitted by larval stage Trombiculid mites (chiggers), whose primary hosts are small mammals. The phylogenomics of <em>O. tsutsugamushi</em> in chiggers, small mammals and humans remains poorly understood. To combat the limitations imposed by the low relative quantities of pathogen DNA in typical <em>O. tsutsugamushi</em> clinical and ecological samples, along with the technical, safety and cost limitations of cell culture, a novel probe-based target enrichment sequencing protocol was developed. The method was designed to capture variation among conserved genes and facilitate phylogenomic analysis at the scale of population samples. A whole-genome amplification step was incorporated to enhance the efficiency of sequencing by reducing duplication rates. This resulted in on-target capture rates of up to 93% for a diverse set of human, chigger, and rodent samples, with the greatest success rate in samples with real-time PCR Ct values below 35. Analysis of the best-performing samples revealed phylogeographic clustering at local, provincial and international scales. Applying the methodology to a comprehensive set of samples could yield a more complete understanding of the ecology, genomic evolution and population structure of <em>O. tsutsugamushi</em> and other similarly challenging organisms, with potential benefits in the development of diagnostic tests and vaccines.
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