We thank A. Kosmrlj for discussions and code; A. P. Aiden, X. R. Bao, M. Brenner, D. Galas, W. Gosper, A. Jaffer, A. Melnikov, A. Miele, G. Giannoukos, C. Nusbaum, A. J. M. Walhout, L. Wood, and K. Zeldovich for discussions; and L. Gaffney and B. Wong for help with visualization. 
Supported by a Fannie and John Hertz Foundation graduate fellowship, a National Defense Science and Engineering graduate fellowship, an NSF graduate fellowship, the National Space Biomedical Research Institute, and grant no. T32 HG002295 from the National Human Genome Research Institute (NHGRI) (E.L.); i2b2 (Informatics for Integrating Biology and the Bedside), the NIH-supported Center for Biomedical Computing at Brigham and Women s Hospital (L.A.M.); grant no. HG003143 from the NHGRI, and a Keck Foundation distinguished young scholar award (J.D.). Raw and mapped Hi-C sequence data has been deposited at the GEO database (<a href="http://www.ncbi.nlm.nih.gov/geo/">www.ncbi.nlm.nih.gov/geo/</a>), accession no. GSE18199. Additional visualizations are available at <a href="http://hic.umassmed.edu">http://hic.umassmed.edu</a>.

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A provisional patent on the Hi-C method (no. 61/100,151) is under review.

We present a method of studying the 3-dimensional architecture of the genome by mapping chromatin interactions in an unbiased, genome-wide manner. The most critical experimental step what sets this technology apart from previous work - is the incorporation of biotinylated nucleotides at the restriction ends of crosslinked fragments before blunt-end ligation. Performing this step successfully enables deep sequencing of all ligation junctions, and gives Hi-C its scope and power. The number of reads will ultimately determine the resolution of the interaction maps. Here, a 1 Mb interaction map for the human genome is presented using ~30 million alignable reads. In order to increase 'all-purpose' resolution by a factor of n, the number of reads must be increased by a factor of n2. The Hi-C technique may be readily combined with other techniques, such as hybrid capture after library generation (to target specific parts of the genome) and chromatin immunoprecipitation after ligation (to examine the chromatin environment of regions associated with specific proteins).

Erratum:

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Protease inhibitors</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P8340-5ml</td>
<td>Step 1.2</td>
</tr>
<tr>
<td>biotin-14-dCTP</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>19518-018</td>
<td>Step 1.6</td>
</tr>
<tr>
<td>Klenow</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>M0210</td>
<td>Steps 1.6 and 2.2</td>
</tr>
<tr>
<td>T4 DNA ligase</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15224</td>
<td>Step 1.9</td>
</tr>
<tr>
<td>T4 DNA polymerase</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>M0203</td>
<td>Steps 1.17 and 2.2</td>
</tr>
<tr>
<td>10x ligation buffer</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>B0202</td>
<td>Steps 2.2 and 3.4</td>
</tr>
<tr>
<td>T4 PNK</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>M0201</td>
<td>Step 2.2</td>
</tr>
<tr>
<td>Klenow (exo-)</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>M0212</td>
<td>Step 2.3</td>
</tr>
<tr>
<td>Dynabeads MyOne Streptavin C1 Beads</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>650.01</td>
<td>Step 3.2</td>
</tr>
<tr>
<td>T4 DNA ligase HC</td>
<td>&nbsp;</td>
<td>Enzymatics</td>
<td>L603-HC-L</td>
<td>Step 3.5</td>
</tr>
<tr>
<td>Phusion HF mastermix</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>F531</td>
<td>Step 3.8</td>
</tr>
<tr>
<td>Ampure beads</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>A2915</td>
<td>Step 3.9</td>
</tr>		</tbody>
		</table>
		</div>

hi-c: a method to study the three-dimensional architecture of genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

Watch this Scientific Journal Video about Hi-C: A Method to Study the Three-dimensional Architecture of Genomes. at JoVE.com

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

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407.8K Views.  University of Massachusetts Medical School. The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale. 