ABSTRACT The detection of chromosomal aberrations is a frontline diagnostic for the spectrum of blood neoplasms. Chromosomal aberrations, such as translocations, inversions, deletions and insertions, have been historically identified using cytogenetic methods or more recently through application of long read sequencing or optical mapping technologies. These methods have been less applicable in solid tumor research and diagnostics because they require either viable cells or high-molecular weight DNA. The vast majority of solid tumor biopsies are stored in formalin-fixed paraffin-embedded (FFPE) blocks, a process that highly fragments genomic DNA. In this proposal we describe a low-cost and scalable method compatible with FFPE tissue that enables the detection of chromosomal aberration using proximity ligation sequencing. Proximity ligation methods such as chromosome conformation capture (3C) and Hi-C can be used to order and orient segments of genomes, reconstructing end-to-end chromosome sequences. When a sequence deviates from the expected order or orientation, such as is in the case of chromosomal aberrations, the sequence appears as an obvious off-diagonal signal on a Hi-C heatmap, making identification of chromosomal abnormalities an automatable process. 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 FFPE Hi-C protocol amenable to multiwell plate handling, 2) building a robust automated platform to reproducibly call chromosome aberrations from Hi-C data, and 3) proving the validity and reproducibility of these methods on real world sample. The result of these efforts will be a new cancer cytogenetics methodology called Karyotyping by SequencingTM (KBS).

NARRATIVE For decades chromosomal aberrations have been useful biomarkers for mechanistic investigations of, diagnosis, and treatment decision making for liquid tumors. The utility of chromosomal aberration in diagnosis and stratification of solid tumor treatment options is limited by the fact that virtually all solid tumor samples are stored as formalin-fixed paraffin embedded (FFPE) tissue. In this proposal we describe a scalable proximity ligation sequencing method and analysis platform that can overcome limitations presented by the highly fragmented DNA found in FFPE tissue to identify chromosomal aberrations in solid tumors in FFPE blocks, enabling research and diagnostic applications.