ABSTRACT Cytogenetic testing is a cornerstone of clinical genetics diagnostics. However, even with state-of-the-art methodologies, available clinical cytogenetic methods have a non-overlapping series of limitations. The resolution of karyotyping is typically estimated to be in the 5-10 megabase pair (Mbp) range, limiting the ability to definitively localize breakpoints. Fluorescence in situ hybridization (FISH) methods overcome some of these limitations, but allow only one or in some cases a few loci to be interrogated at a time. Chromosomal microarray analysis (CMA) offers important advantages over karyotyping and FISH, including applicability to virtually any tissue, increased resolution, and semiautomated interpretation, but is unable to call balanced translocations, inversions, complex rearrangements, and changes in ploidy. Thus, several complementary tests are typically used in parallel or in sequence and this greatly inflates the costs and turnaround times of cytogenetic testing. Proximity ligation methods such as chromosome conformation capture (3C, Hi-C) can be used to measure physical and genetic distance between all pairs of loci simultaneously on a full-chromosome scale. This property has been primarily applied to reconstructing end-to-end chromosome sequences for animals and plants. However, this technology is extremely well suited to the detection of chromosomal aberrations using relatively simple and ubiquitous sequencing tools and scalable computational analytics. We propose to apply proximity ligation as a cytogenomic method to detect the breadth of chromosomal aberrations at high resolution and low cost. This proposal outlines a path to a commercially available product and service, which will establish a highly validated method for use in research and eventually in a diagnostic setting. This will be accomplished by 1) designing an easy to use Hi-C protocol amenable to multiwell plate handling, 2) building a robust automated platform to reproducibly call chromosomal aberrations from Hi-C data, and 3) proving the validity and reproducibility of these methods on real world samples. The resulting kit and software product will be a new cytogenomic method called Karyotyping by SequencingTM (KBS).