ABSTRACT Emerging genomic medicine initiatives are poised to impact prenatal and pediatric diagnostics, including assessment of fetal structural anomalies (FSAs) from amniocentesis and the rapid adoption of non-invasive prenatal screening (NIPS). Indeed, the rapid decrease in whole-genome sequencing (WGS) costs and the improved sophistication of analytic genomics methods has brought prenatal screening to a critical inflection point. Ongoing studies find improved diagnostic yields from WGS and whole exome sequencing (WES) over conventional karyotype and microarray (CMA); however, the technical, analytical, and interpretative challenges presented by structural variants (SVs) continue to confound sequence-based diagnostics. The field lacks standardized methods to interpret SVs from WGS and WES, yet these variants underlie a significant fraction of prenatal diagnoses, particularly for high risk fetuses with multiple congenital anomalies (MCAs). Our clinical SV program was initially formed to nucleate expertise in technology and algorithm development, variant interpretation, maternal-fetal medicine, and large-scale references to explore the impact of high-resolution SV detection in prenatal diagnostics. This resubmission builds upon the methods, resources, and discoveries from those studies to establish uniform approaches to jointly discover and interpret SVs, initially from amniocentesis and ultimately using non-invasive methods. We will focus on MCAs as exemplars of genomic diagnostics in severe clinical referrals. The discoveries from our initial funding period collectively suggest several critical advances could transform prenatal screening and genetic diagnostics, and we directly address three major barriers to these advances in this renewal: (1) Diagnostic yields from WES in FSAs are highly variable due to inconsistent methods and limited sensitivity to capture SVs. Aim 1 will benchmark diagnostic yields from WGS in MCA cases using our standardized open-source pipelines. (2) The genes contributing to the most severe fetal anomalies in humans remain largely unknown, as studies of MCAs have mostly been restricted to small cohorts and low-resolution CMA methods. Aim 2 will aggregate these severe fetal anomalies and perform uniform variant detection and joint association analyses of short variants and SVs from WES and WGS in FSA trios across multiple consortia, which we will compare to population-scale aggregated controls. (3) NIPS is ultra-low resolution and fails to capture most causal variant classes underlying MCAs. Aim 3 will benchmark an innovative approach to detect coding mutations and SVs from cffDNA, comparing yields to current NIPS and amniocentesis as the standard-of-care. Our team will thus leverage complementary expertise, novel methods, and unique patient resources to advance routine genomic screening in prenatal diagnostics.