Multilocus sequence typing anchors molecular epidemiology, but traditional frameworks require centrally curated allele catalogues. For emerging and uncultivable eukaryotic parasites, maintaining these databases is impractical, leaving surveillance reliant on fragmented, assay-specific scripts. PyEuk eliminates this bottleneck by providing an open, catalogue-free suite that calls microhaplotypes directly from sequence differences relative to a reference within data-defined genomic windows. When amplicon coordinates are uncharacterized or unpublished, PyEuk reconstructs target panels de novo from raw read coverage peaks mapped to a draft assembly. Across benchmark cohorts spanning Cyclospora cayetanensis and Plasmodium vivax, PyEuk recovers epidemiological structure established by tracebacks, geography, and clinical recurrence without organism-specific tuning. In foodborne outbreaks, it resolves independent transmission chains using either curated or de novo panels and scales to national surveillance archives exceeding 8,000 isolates. In P. vivax malaria, its weighted identity-by-state distance separates continental lineages, discriminates liver-stage relapses from reinfections, and delineates transmission clusters. Rather than forcing an arbitrary partition on continuous variation, PyEuk evaluates bootstrap stability, reporting supported cluster count ranges alongside reproducible transmission cores. PyEuk provides a portable, reproducible foundation for eukaryotic pathogen surveillance.
Kosakovsky Pond, S. L., Callan, D., Nekrutenko, A.
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