ABSTRACT The Undiagnosed Diseases Network (UDN) has increased access for patients with undiagnosed diseases to the nation’s leading clinicians and scientists. The extension of Phase II of the Center for Undiagnosed Diseases at Stanford will facilitate the continuation of UDN efforts toward sustainability, refinement of methods, and integration with regular clinical practice. Here, we propose a program of study that will (1) facilitate accurate diagnosis of patients with undiagnosed diseases; (2) improve diagnostic rates through novel approaches to data analysis and integration; and (3) explore underlying mechanisms of disease to accelerate therapeutic drug discovery. In Aim 1, we propose to complete clinical evaluations and associated work for participants enrolled in the UDN at Stanford. This will include pre-visit chart review and genetic counseling followed by an individualized visit during which standardized phenotypic and environmental data are collected. Biosamples facilitate genomic, multi-omic, and cellular evaluation of disease. Expansion of fibroblasts and, in selected cases, generation of induced Pluripotent Stem Cell (iPSC) lines facilitates scientific investigation of the underlying diseases. We propose to continue implementation of novel methods in areas of high potential to increase diagnostic yield. This includes algorithms for the detection of small genomic insertions and deletions, structural variation and differentially methylated regions. We will continue evaluation of long-read sequencing technologies. We will apply novel computational approaches for systematic integration of genomic, transcriptomic, metabolomic, and phenotypic data with the entire medical literature to improve diagnostic yield. We also propose to facilitate diagnosis through enhanced cellular and model organisms phenotyping. We will implement immunomic approaches for undiagnosed cases with suspected immune mechanisms. In Aim 2, we will prepare for the transition to the Phase III model of the UDN. This will include preparing both samples and data for archiving, together with continuing network activities to enhance the value of the UDN beyond the funding period. We will continue our efforts to seek diagnosis using available resources for all participants.