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3D Chromatin Conformation Capture Methods

There are several different techniques that provide targeted or genome-scale chromatin conformation (3D) information.  Generally, these techniques employ DNA-crosslinking (typically with formaldehyde), digestion, ligation, capture and sequencing to visualize the physical proximity between chromatin regions. 

The scale of library complexity and sequencing depth on genome-scale 3D techniques can define TADs (topologically-associated domains), LADs (lamina-associated domains) and chromatin loops. These techniques are also used in genome assembly by orienting and anchoring fragmented sequence contigs into complete, chromosome-scale genomic scaffolds.

At this time, the CGI does not offer the technique due to variable sample inputs, we can provide recommended protocols, experimental design and can perform QC, library preparation and sequencing for your prepared sample(s).

For Illumina-based methods (Hi-C, TELL-seq, Omni-C, Link Prep, Micro-C), the CGI can prepare and sequence libraries with a recommended depth of sequencing 400M PE reads (600M-800M if your library has enough complexity and you are aiming for gene-level domains). We recommend a screening run on a MiSeq or i100+ to determine if your library has high complexity, and a high percentage of no-dup valid read pairs.

For LRS-based techniques (Pore-C and CiFi) the CGI can perform library preparation, QC and sequencing on either the ONT PromethION or PacBio Revio, as appropriate. A single Flow Cell/Smart Cell is recommended for a genome size of 3GB, but coverage will depend on the quality of the sequencing run which can vary depending on the sample type, species and proximity-ligation prepared DNA.

Hi-C begins with cross-linking and DNA digestion with a specific restriction enzyme. Post-digestion the 5’ overhangs are filled in, incorporating biotinylated nucleotides. After blunt-end ligation and reversal of the cross-links, the biotin tag at the center of the ligation junction in the chimeric DNA strand are pulled down using streptavidin beads. This results in a sample that is primarily composed of hybrid strands representing informative contacts. Purified samples are used to produce sequencing libraries for NGS on an Illumina platform. 

Ideal for smaller genomes (such as yeast and bacteria), TELL-seq is a method developed by Universal Sequencing that produces genome-scale 3D contact information by leveraging a dense solution to enable efficient DNA partitioning and co-barcoding in a single PCR tube.

Micro-C is the same approach as in Hi-C but instead of a restriction enzyme, this method uses a micrococcal nuclease (MNase) for nucleosome resolution. 

Omni-C is the same approach as in Hi-C but instead of a restriction enzyme or MNase, this method uses a sequence-independent endonuclease to provide uniform coverage across the genome. 

LinkPrep uses tagmentation (Tn5 transposase) for fragmentation and adaptor integration of chromatin instead of restriction enzymes to provide uniform coverage across the genome. This method can be performed in a single-day.

Both Pore-C and CiFi are long-read based genome-wide conformation capture techniques that utilize proximity ligation and either an endonuclease or restriction enzyme to detect and visualize proximity between chromatin regions. Pore-C is adapted for LRS on Oxford Nanopore platforms (particularly the PromethION) while CiFi is adapted for LRS on the PacBio Revio.