ABSTRACT Mendelian conditions, particularly those presenting during childhood, are a major disease burden causing suffering and taxing the healthcare system. Clinical approaches including exome sequencing have led to a rapid increase in the number of conditions with known genetic causes and new treatment options for many rare diseases, but are restricted to finding small coding and splice junction variants, missing most non-coding variants as well as structural and copy number variants. This challenges interpretation due to the vast number of variants that must be analyzed for possibly causing a disease. To accelerate the pace of Mendelian disease gene discovery and clinical implementation, we propose a Pediatric Mendelian Genomics Research Center (MGRC), leveraging the broad pediatric clinical and research expertise of Children’s National Hospital and Research Institute in a partnership with Invitae’s expertise in providing comprehensive and affordable genetic testing. Our Center will unite world class experts combining basic and translational research with innovative approaches to phenotyping, variant identification and functional investigation of both coding and non-coding sequence changes with the goals of discovering novel Mendelian gene variations and identifying variants not detected on current sequencing pipelines, disambiguating uncertain variants into disease-causing versus benign categorizations, and sharing information by working collaboratively with the MGRC community. To answer these challenges, this proposal will address the following Specific Aims: Aim 1: Identify novel causes of Mendelian conditions - Discover: Our center will enroll patients with likely Mendelian diseases and previously non-diagnostic tests (2,600 samples per year) then systematically re- analyze whole genomes augmented with long read sequencing, optical mapping, and RNA-seq. Aim 2: Reclassify uncertain variants and investigate the mechanisms of undiagnosed Mendelian conditions - Disambiguate: Uncertain variants and candidate genes will be further investigated using whole transcriptome analysis, RNA-seq, CRE-seq, and functional modeling. Aim 3: Communicate research results to enable translational research on new and rare Mendelian conditions - Disseminate: Our center is committed to data sharing and dissemination and will ensure that data is shared with the entire MGRC community through the data coordinating center. Through our industry partnership, clinically valid pipelines will be rapidly scaled for clinical implementation globally. Our overall approach provides an efficient and direct path to diagnosis for patients affected with undiagnosed Mendelian conditions, promotes gene discovery and reclassification of Variants of Uncertain Significance through a combination of innovative approaches, and will allow individuals, families and healthcare providers to improve the management of disease.