Understanding the physical processes behind DNA-DNA proximity ligation assays

In the last decade, DNA-DNA proximity ligation assays opened up powerful new ways to study the 3D organization of genomes, and have rapidly become a mainstay experimental technology. Yet many aspects of these experiments are still poorly understood. We study the inner workings of DNA-DNA proximity ligation assays by means of numerical experiments and theoretical modeling. Chromosomes are modeled at nucleosome resolution and evolved in time via molecular dynamics. A virtual Hi-C experiment is performed, in silico, reproducing the different steps of the Hi-C protocol. The virtual Hi-C protocol includes crosslinking of chromatin to an underlying proteic matrix, the action of restriction enzymes cutting DNA, and the subsequent ligation of DNA open ends which are found in proximity. The protocol is performed on ensembles of different structures as well as individual structures. Simulations enable the construction of ligation maps for the virtual Hi-C experiments and the calculation of ligation probabilities as functions of both genomic and Euclidean distance, as well as statistics of self-ligations or cyclization. Our results allow close examination of the features and pitfalls of the Hi-C methodology, as well as the ensuing data processing. Additionally, the methods developed allow characterizing how the many variables present in the experimental protocol affect the quality of results.