Introduction of Some New Technologies (3)

November 13, 2018

Hi-C method is based on Chromosome Conformation Capture, in that chromatin is crosslinked with formaldehyde, then digested, and re-ligated in such a way that only DNA fragments that are covalently linked together form ligation products. The ligation products contain the information of not only where they originated from in the genomic sequence but also where they reside, physically, in the 3D organization of the genome. In Hi-C, a biotin-labeled nucleotide is incorporated at the ligation junction, making it possible to enrich for chimeric DNA ligation junctions when modifying the DNA molecules for deep sequencing. The compatibility of Hi-C with next generation sequencing platforms makes it possible to detect chromatin interactions on an unprecedented scale. This advance gives Hi-C the power to both explore the chromatin biophysics as well as the implications of chromatin structure in the biological functions of the nucleus. A massively parallel survey of chromatin interaction provides the previously missing dimension of spatial context to other genomic studies. This spatial context will provide a new perspective to studies of chromatin and its role in genome regulation in normal conditions and in disease.

Figure 1. Schematic workflow of Hi-C sequencing.

Sequencing Strategy and Recommended Depth

Illumina HiSeq PE150 sequencing with depth of coverage ≥ 30x

Data Analysis

• Quality control
• Hi-C marking and ligation efficiency estimation
• Analysis of the sequenced reads
• The heatmap of chromatin interactions
• Different ways of analyzing the data to reveal various levels of genome organization.
• Preferential association between particular chromosome pairs.
• New insights were gained into chromatin folding at the megabase scale.

Sample Requirements

• Cell: 5×107-1×108, ensure experimental reproducibility
• DNA amount: 15-20ug
• DNA concentration: ≥ 20 ng/μl
• Purity: OD260/280 = 1.8 - 2.0, without degradation or RNA contamination

Key Features and Advantages

1. Allows detection of long-range DNA interactions
2. High-throughput method
3. A single sample can achieve auxiliary genome assembly
4. High accuracy
5. Short cycle, cost-effective