Undiagnosed diseases are likely to be determined by genetic, environmental, and developmental factors. While some undiagnosed diseases will represent novel rare genetic syndromes with monogenic or oligogenic etiologies and others will reflect rare manifestations of known diseases, many are likely to result from a more analytically challenging combination of multiple genetic, environmental, and developmental factors. Whole exome and whole genome sequencing are powerful tools with which to ascertain the genetic contributions to undiagnosed diseases; however, these methods alone are unlikely to elucidate the basis for many, if not most, undiagnosed diseases. The overarching approach we have taken in this Continuation application is to extend the methodologies that we have established in earlier phases of the UDN and to add key components specifically designed to continue to optimize case ascertainment and phenotyping (Aim 1), to refine integrated multidisciplinary disease diagnosis (Aim 2) and to directly support the deployment and further optimization of a fully sustainable UDN model (Aim 3). These specific elements include: • Broadening of the initial case assessment population (including enrichment for health disparity populations) to enable better definition of the difficult-to-diagnose patient who might benefit from UDN approaches; • Development of quantitative triage tools for difficult-to-diagnose patients enabling continued optimization of case selection and the utility of individual techniques; • Deployment of innovative new sequence interpretation pipelines merging the best techniques and personnel from across the entire Boston ecosystem (with additional engagement from BUMC and the Broad Institute); • Broader integration of functional genomics tools in the initial assessment of cases entering the UDN; • Formal deployment and quantitative evaluation of a Boston-wide UDN center funded through active commercial contracts with payers and DHHS (and cross-subsidized through other non-UDN genetics programs in our system, such as Brigham Genomic Medicine); and • Prospective design of all of these elements to allow dissemination of successful components across the entire UDN and beyond. These efforts will maximize the yield of UDN resources while progressively broadening access in a fully sustainable (both practically and financially) model.