Ivo ElliottNeeranuch ThangnimitchokMariateresa de CesarePiyada LinsuwanonDaniel H. ParisNicholas P.J. DayPaul N. NewtonRory BowdenElizabeth M. BattyThe Wellcome Centre for Human GeneticsWalter and Eliza Hall Institute of Medical ResearchUniversitat BaselSwiss Tropical and Public Health Institute (Swiss TPH)Armed Forces Research Institute of Medical Sciences, ThailandMahosot Hospital, LaoMahidol UniversityNuffield Department of Medicine2022-08-042022-08-042021-07-01Infection, Genetics and Evolution. Vol.91, (2021)15677257156713482-s2.0-85103408642https://repository.li.mahidol.ac.th/handle/20.500.14594/75636Scrub typhus is a febrile disease caused by Orientia tsutsugamushi, transmitted by larval stage Trombiculid mites (chiggers), whose primary hosts are small mammals. The phylogenomics of O. tsutsugamushi in chiggers, small mammals and humans remains poorly understood. To combat the limitations imposed by the low relative quantities of pathogen DNA in typical O. tsutsugamushi 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 O. tsutsugamushi and other similarly challenging organisms, with potential benefits in the development of diagnostic tests and vaccines.Mahidol UniversityAgricultural and Biological SciencesBiochemistry, Genetics and Molecular BiologyImmunology and MicrobiologyMedicineArticleSCOPUS10.1016/j.meegid.2021.104818