SUMMARY Our program will develop a comprehensive genomic resource to characterize novel causes of unexplained recurrent pregnancy loss (RPL). We access large clinical service for RPL to prospectively procure carefully phenotyped trios with fetal cells available from spontaneous pregnancy losses along with both parents’ DNA and paternal germ cells. In addition, we leverage our large health-system wide Genomic Answers for Kids (GA4K) program developed for understanding pediatric rare disease currently with over 5500 trios enrolled enriched for prior pregnancy losses. Using these rich data and clinical resources, we aim to extend genetic testing workflow by integrating our clinically deployed long-read genome sequencing (HiFi-GS) pipeline using PacBio Revio allowing simultaneous DNA (cytosine) methylation in long, contiguous read (‘5-base HiFi-GS’) for the identification of underexplored genomic variants and hidden functional features. We couple these third- generation genomic analyses in trios with contemporary electronic medical records (EHR) and clinical and population-based biobank resources (UK Biobank). The data integration will leverage and extend our preliminary findings to test a central hypothesis that unexplained RPL is due to inherited or de novo changes in full genome sequences. Specifically, we propose in Aim 1 to leverage GA4K’s and our hospital’s detailed clinical and molecular diagnostic data together with dynamic access to EHR and extract pregnancy history to classify molecular variation linked to RPL. We validate these links in thousands of RPL cases observed in UK Biobank by identifying deleterious variants in the available exome sequencing data. In Aim 2 we utilize our high- throughput 5-base HiFi-GS platform to consolidate all known molecular tests for genetic disease into a single sequencing run for the identification of putative variants and complex DNA changes missed by clinical microarray and unmeasured by standard GS. We will assess 100 unsolved rare disease families from GA4K with history of pregnancy loss and 100 RPL trios. This 5-HiFi-GS resource will also allow unbiased information on skewed X- inactivation and imprinting defects in fetal DNA, with poorly understood impacts on fetal viability. Finally, in Aim 3 we apply CpG methylation detection by 5-HiFi-GS in paternal germ cells (sperm) contrasting samples derived from male partner with RPL with those who have proven paternity to compare levels of gonadal mosaicism for genetic variants as well as epigenomic transmission observable in 5-HiFi-GS from paternal blood and sperm together with fetal DNA. Expected results entail compendium of high-quality full RPL genomes to spark community-wide research with novel insight to dozens of RPL genes linked to genetic disease and impacting fetal viability.