show Abstracthide AbstractA crucial aspect of validating any clinical assay is the availability of well-characterized samples to assess the specificity and sensitivity of the methodology being tested. However, due to logistical constraints, the biosafety requirements of the specimen studied, or geographic diversity, many laboratories struggle to access adequate clinical specimens or isolates. Consequently, a diverse and well-characterized dataset for standardized assay validation is needed. The Wadsworth Center, the New York State Department of Health's laboratory, has been performing whole-genome sequencing (WGS) on every tuberculosis (TB) case in the state since 2016, along with comprehensive phenotypic drug susceptibility testing (DST) of all drug-resistant isolates. This work has resulted in a large collection of fully characterized clinical Mycobacterium tuberculosis complex isolates, complete with whole-genome sequencing data and paired phenotypic drug resistance profiles. To support the TB community, we have compiled a comprehensive dataset from 50 of these well-characterized isolates, sequenced using the Illumina MiSeq platform. This dataset has been curated to be inclusive of a broad range of lineage diversity, drug resistance types, and resistance-associated gene mutations. This dataset is now available to the community for method development and bioinformatics pipeline validation, serving as a valuable resource to advance research and enhance clinical WGS assays.