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
[edit | edit source]Useful background
[edit | edit source]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
[edit | edit source]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
[edit | edit source]Through these techniques we can study from single enhancer-gene interactions up to the global principles of genome organisation.
Inputs and outputs
[edit | edit source]Inputs
[edit | edit source]- Raw hybrid or paired reads
Outputs
[edit | edit source]- Interaction frequencies between chosen regions
- Topological domains and other physical chromatin structures
Experimental design
[edit | edit source]The resolution at which a 3C-based experiment can be analysed is determined by two factors:
- The primary restriction enzyme used to fragment the DNA
- 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.[1]
Typical steps in the method
[edit | edit source]- 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
[edit | edit source]Normalisation and correction for contact biases can be done using a number of published tools.
Workflows
[edit | edit source]Example galaxy workflow
[edit | edit source]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.
Example command line workflow
[edit | edit source]Discussion
[edit | edit source]POV discussion about the method.