Next Generation Sequencing (NGS)/Chromatin conformation capture

Chromatin conformation capture (3C) technologies are used to discover which regions of DNA are physically co-located in space, through ligation and sequencing the hybridised product. The original 3C experiments counted the level of interaction between two regions of interest and subsequent assays using NGS have scaled this up to measure genome-wide contacts.

Overview

Useful background

An understanding of the components of chromatin (DNA along with everything bound to it) and the cell cycle help understand what causes and effects the interactions measured by chromation conformation capture.

Objective

Interaction frequencies between either two selected fragments (3C), a bait region against all other fragments (4C), an all-against-all sampling of a given region (5C) or genome-wide assesment of interaction frequencies (Hi-C).

Biological questions

Through these techniques we can study from single enhancer-gene interactions up to the global principles of genome organisation.

Inputs and outputs

Inputs

• Raw hybrid or paired reads

Outputs

• Interaction frequencies between chosen regions
• Topological domains and other physical chromatin structures

Experimental design

Overview of assay procedure for chromosome conformation capture methods 3C, 4C and 5C.

The resolution at which a 3C-based experiment can be analysed is determined by two factors:

1. The primary restriction enzyme used to fragment the DNA
2. The sequencing depth over the target region

A 6-cutter restriction enzyme (such as HindIII) limits the resolution to fragments with mean size of around 3.5 kb, whereas a 4-cutter such as DpnI reduces fragment size by around an order of magnitude.

Typical steps in the method

• Map reads to reference genome. In the case of paired reads (Hi-C), reads should be mapped independently and not using an alignment program with "paired-end mode" or similar. This mode will likely impose constraints on the distance between mapped paired reads, when in this assay even contacts many megabases away (and in trans across chromosomes) still give useful information.

Next steps

Normalisation and correction for contact biases can be done using a number of published tools.

Workflows

Example galaxy workflow

Link to an example galaxy workflow for for the method (including example datasets) on a given galaxy instance or to the XML document describing the workflow.

Discussion

POV discussion about the method.