Chromosomal aberration detection in FFPE tissue using proximity ligation sequencing

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).