Iteratively improving Hi-C experiments one step at a time

Abstract

(250 words)The 3D organization of eukaryotic chromosomes affects key processes such as gene expression, DNA replication, cell division, and response to DNA damage. The genome-wide chromosome conformation capture (Hi-C) approach can characterize the landscape of 3D genome organization by measuring interaction frequencies between all genomic regions. Hi-C protocol improvements and rapid advances in DNA sequencing power have made Hi-C useful to diverse biological systems, not only to elucidate the role of 3D genome structure in proper cellular function, but also to characterize genomic rearrangements, assemble new genomes, and consider chromatin interactions as potential biomarkers for diseases. Yet, the Hi-C protocol is still complex and subject to variations at numerous steps that can affect the resulting data. Thus, there is still a need for better understanding and control of factors that contribute to Hi-C experiment success and data quality. Here, we evaluate recently proposed Hi-C protocol modifications as well as often overlooked variables in sample preparation and examine their effects on Hi-C data quality. We examine artifacts that can occur during Hi-C library preparation, including microhomology-based artificial template copying and chimera formation that can add noise to the downstream data. Exploring the mechanisms underlying Hi-C artifacts pinpoints steps that should be further optimized in the future. To improve the utility of Hi-C in characterizing the 3D genome of specialized populations of cells or small samples of primary tissue, we identify steps prone to DNA loss which should be optimized to adapt Hi-C to lower cell numbers.Keywords: Hi-C; chromosome conformation capture; 3D genome structure; high throughput sequencing Highlights: 3 to 5 bullet points (maximum 85 characters, including spaces, per bullet point)  Variability in Hi-C libraries can arise from early steps of cell preparation  Hi-C 2.0 changes to interaction capture steps also benefit 6-cutter libraries  Artificial molecule fusions can arise during end repair and PCR, increasing noise 

Radiation-induced DNA damage and repair effects on 3D genome organization

“…Hi-C. To prepare the Hi-C libraries, at the appropriate timepoints, Hi-C experiments were performed according to the protocol detailed in Golloshi et al 66 . Briefly, 5-20 million cells were fixed with 1% formaldehyde, suspended in cell lysis buffer for permeabilization, and homogenized by douncing.…”

In situ fixation and subsequent collection of cultured endothelial cells in a shear flow

“…, flasks, dishes, welled plates) are fixed in situ and removed from the culture dish using a cell scraper [[8], [9], [10]]. Although scraping can effectively remove adherent cells, this technique introduces variation of the external force on the cell [11]. Moreover, the scraping process affects cells differently based on two parameters: (1) the mechanical stiffness of the cell line; and, (2) the force used to remove the cells from the growth surface, which may vary substantially between experimenters and between experiments.…”

Inferring Chromosome Radial Organization from Hi-C Data

“…Hi-C experiments capture spatial genome organization by measuring the interaction frequency between different genomic fragments, yielding information about both intra-chromosomal and inter-chromosomal interactions. Inter-chromosomal interactions generally occur much less frequently than intra-chromosomal interactions, and true interactions are mixed in with noise that arises from random background ligation [57,30]. Despite this inherent noise and sometimes low signal, the interactions between chromosomes also contain information that reflects the radial organization of the chromosome territories inside the nucleus.…”

“…In contrast, genome wide chromosome conformation capture (Hi-C) approaches are being applied to characterize the 3D genome structure of rapidly increasing numbers of cell types and conditions for the past decade [1,29,30,31]. This Hi-C technique and its variants capture a snapshot of pairwise chromosomal interactions ranging from specific enhancerpromoter interactions [32] to large scale inter-chromosomal interactions.…”