3D genome mapping
Genome-wide proximity ligation: restriction digestion of crosslinked chromatin, biotin fill-in, ligation inside intact nuclei (in situ Hi-C), biotin pull-down and paired-end sequencing. Gives an all-against-all contact matrix whose resolution is set by enzyme and depth.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Hi-C
Genome-wide proximity ligation: restriction digestion of crosslinked chromatin, biotin fill-in, ligation inside intact nuclei (in situ Hi-C), biotin pull-down and paired-end sequencing. Gives an all-against-all contact matrix whose resolution is set by enzyme and depth.
How it differs from the standard method
- Four-cutters (DpnII, MboI) give finer and more even maps than six-cutters (HindIII).
- Kilobase-scale loops need billions of read pairs genome-wide; domains need far fewer.
- QC: valid-pair fraction, cis/trans ratio, long-range cis share, duplicates, and the P(s) curve.
1Turn closeness in the nucleus into a sequence you can read
Chromosome conformation capture methods ask which pieces of the genome sit next to each other inside the nucleus. Formaldehyde freezes the folded chromatin in place.
All against all
A restriction enzyme cuts the fixed chromatin, the ends are filled in with a biotinylated nucleotide and ligated inside intact nuclei, and biotin pull-down enriches the junctions. Every read pair is one contact, anywhere in the genome.
Down to the nucleosome
MNase replaces the restriction enzyme and digests the chromatin into single nucleosomes. Fragments are then the size of a nucleosome rather than set by where the enzyme's motif happens to fall, so the map can show contacts a few kilobases apart.
One against all
After a first digestion and ligation, a second digestion and circularisation leave every partner of one chosen viewpoint in a small circle. Inverse PCR from primers on the viewpoint amplifies all of them, which gives a deep, cheap profile of one locus.
2What the map shows
Contact frequency falls steeply with distance along the chromosome, roughly as distance to the power −1 over hundreds of kilobases, so the map is brightest along its diagonal. On top of that decay sit squares of enriched contact (topologically associating domains) separated by boundaries where contacts are insulated, often bound by CTCF and cohesin.
3Where it goes wrong
- Without crosslinking, cut fragments drift apart and ligate at random: no decay with distance, most pairs in trans.
- Ligation in a dilute solution after dissolving the nuclei adds random intermolecular junctions; in situ ligation inside nuclei keeps them rare.
- Biotin left on unligated ends pulls down 'dangling end' fragments with no junction in them.
- Bins finer than the data support give a speckled map: a common rule is that 80% of bins need at least 1,000 contacts at the chosen resolution.
- A six-cutter gives few, uneven fragments; four-cutters (or MNase) give finer, more even coverage.
- Unbalanced matrices make poorly covered bins look like boundaries; iterative correction (ICE) or similar balancing is standard.
- In 4C, all reads start at the same primer, so duplicates cannot be removed: low template gives a profile of a few products copied thousands of times.
Key terms
Practise these as flashcards →Counts of read pairs between every pair of genomic bins; symmetric about the diagonal.
Topologically associating domain: a region whose loci contact each other more than they contact their neighbours.
Mean contact frequency across a window sliding along the diagonal; its minima mark domain boundaries.
A focal enrichment of contacts between two distant loci, often anchored by convergent CTCF sites.
Contact probability against genomic distance; its slope summarises chromatin compaction.
A read pair from a true ligation junction between two different fragments, after removing dangling ends, self-circles and duplicates.
Share of valid pairs within one chromosome versus between chromosomes; high trans signals random ligation.
In 4C, the restriction fragment whose contacts are amplified by inverse PCR.
When to use it
- Hi-C to map compartments, domains and loops genome-wide, or to scaffold a genome assembly.
- Micro-C when fine structure matters: promoter–enhancer contacts and loops a few kilobases apart.
- 4C to profile one locus deeply and cheaply across many samples or perturbations.
Compared with alternatives
DNA FISH measures distances in single cells directly and checks whether a contact seen in a population map is frequent.
Shows where CTCF and cohesin bind, which explains many of the boundaries and loop anchors.
Identifies the open promoters and enhancers that loops connect.
Assembly and structural variants that can masquerade as contacts in a Hi-C map.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Genome-wide proximity ligation: restriction digestion of crosslinked chromatin, biotin fill-in, ligation inside intact nuclei (in situ Hi-C), biotin pull-down and paired-end sequencing. Gives an all-against-all contact matrix whose resolution is set by enzyme and depth.
- Four-cutters (DpnII, MboI) give finer and more even maps than six-cutters (HindIII).
- Kilobase-scale loops need billions of read pairs genome-wide; domains need far fewer.
- QC: valid-pair fraction, cis/trans ratio, long-range cis share, duplicates, and the P(s) curve.