Hi-C (genomic analysis technique)

Hi-C is a high-throughput genomic and epigenomic technique to capture chromatin conformation (3C). In general, Hi-C is considered as a derivative of a series of chromosome conformation capture technologies, including but not limited to 3C (chromosome conformation capture), 4C (chromosome conformation capture-on-chip/circular chromosome conformation capture), and 5C (chromosome conformation capture carbon copy). Hi-C comprehensively detects genome-wide chromatin interactions in the cell nucleus by combining 3C and next-generation sequencing (NGS) approaches and has been considered as a qualitative leap in C-technology (chromosome conformation capture-based technologies) development and the beginning of 3D genomics. Similar to the classic 3C technique, Hi-C measures the frequency (as an average over a cell population) at which two DNA fragments physically associate in 3D space, linking chromosomal structure directly to the genomic sequence. The general procedure of Hi-C involves first crosslinking chromatin material using formaldehyde. Then, the chromatin is solubilized and fragmented, and interacting loci are re-ligated together to create a genomic library of chimeric DNA molecules. The relative abundance of these chimeras, or ligation products, is correlated to the probability that the respective chromatin fragments interact in 3D space across the cell population. While 3C focuses on the analysis of a set of predetermined genomic loci to offer "one-versus-some" investigations of the conformation of the chromosome regions of interest, Hi-C enables "all-versus-all" interaction profiling by labeling all fragmented chromatin with a biotinylated nucleotide before ligation. As a result, biotin-marked ligation junctions can be purified more efficiently by streptavidin-coated magnetic beads, and chromatin interaction data can be obtained by direct sequencing of the Hi-C library. Analyses of Hi-C data not only reveal the overall genomic structure of chromosomes, but also offer insights into the biophysical properties of chromatin as well as more specific, long-range contacts between distant genomic elements (e.g. between genes and regulatory elements), including how these change over time in response to stimuli. In recent years, Hi-C has found its application in a wide variety of biological fields, including cell growth and division, transcription regulation, fate determination, development, autoimmune disease, and genome evolution. By combining Hi-C data with other datasets such as genome-wide maps of chromatin modifications and gene expression profiles, the functional roles of chromatin conformation in genome regulation and stability can also be delineated.

History At its inception, Hi-C was a low-resolution, high-noise technology that was only capable of describing chromatin interaction regions within a bin size of 1 million base pairs (Mb). The Hi-C library also required several days to construct, and the datasets themselves were low in both output and reproducibility. Nevertheless, Hi-C data offered new insights for chromatin conformation as well as nuclear and genomic architectures, and these prospects motivated scientists to put efforts to modify the technique over the past decade. Between 2012 and 2015, several modifications to the Hi-C protocol have taken place, with 4-cutter digestion or adapted deeper sequencing depth to obtain higher resolution. The use of restriction endonucleases that cut more frequently, or DNaseI and Micrococcal nucleases also significantly increased the resolution of the method. More recently (2017), Belaghzal et al. described a Hi-C 2.0 protocol that was able to achieve kilobase (kb) resolution. The key adaptation to the base protocol was the removal of the SDS solubilization step after digestion to preserve nuclear structure and prevent random ligation between fragmented chromatin by ligation within the intact nuclei, which formed the basis of in situ Hi-C. In 2021, Hi-C 3.0 was described by Lafontaine et al., with higher resolution achieved by enhancing crosslinking with formaldehyde followed by disuccinimidyl glutarate (DSG). While formaldehyde captures the amino and imino groups of both proteins and DNA, the NHS-esters in DSG react with primary amines on proteins and can capture amine-amine interactions. These updates to the base protocol allowed the scientists to look at more detailed conformational structures such as chromosomal compartment and topologically associating domains (TADs), as well as high-resolution conformational features such as DNA loops. To date, a variety of derivatives of Hi-C have already emerged, including in situ Hi-C, low Hi-C, SAFE Hi-C, and Micro-C, with distinctive features related to different aspects of standard Hi-C, but the basic principle has remained the same.

Traditional Hi-C The outline of the classical Hi-C workflow is as follows: cells are cross-linked with formaldehyde; chromatin is digested with a restriction enzyme that generates a 5' overhang; the 5' overhang is filled with biotinylated bases and the resulting blunt-ended DNA is ligated. The ligation products, with biotin at the junction, are selected for using streptavidin and further processed to prepare a library ready for subsequent sequencing efforts. The pairwise interactions that Hi-C can capture across the genome are immense and so it is important to analyze an appropriately large sample size, in order to capture unique interactions that may only be observed in a minority of the general population. To obtain a high complexity library of ligation products that will ensure high resolution and depth of data, a sample of 20–25 million cells is required as input for Hi-C. Primary human samples, which may be available only in fewer cell numbers, could be used for standard Hi-C library preparation with as low as 1–5 million cells. However, using such a low input of cells may be associated with low library complexity which results in a high percentage of duplicate reads during library preparation. Standard Hi-C gives data on pairwise interactions at the resolution of 1 to 10 Mb, requires high sequencing depth and the protocol takes around 7 days to complete.

Formaldehyde cross-linking

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