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5C Sequencing Service: High-Resolution "Many-to-Many" Interaction Mapping

Map complex regulatory neighborhoods with high precision using our 5C Sequencing Service. By combining the specificity of locus-specific primers with the throughput of NGS, 5C (Chromosome Conformation Capture Carbon Copy) generates high-resolution "many-to-many" interaction maps for targeted genomic regions. Ideal for dissecting GWAS intervals, gene clusters, and enhancer-rich loci. RUO.

  • Many-to-Many: Map all interactions within a specific region (e.g., 1–10 Mb).
  • High Resolution: Achieve <5 kb resolution for fine-mapping enhancer contacts.
  • Efficient: Focus sequencing power exclusively on your regions of interest.
  • Custom Design: Expert primer design services for continuous or dispersed targets.
Design Your 5C Panel

Illustration of the 5C Carbon Copy step showing specific primer annealing and ligation.

Overview: Bridging the Gap Between 3C and Hi-C

In the study of 3D genome organization, researchers often face a trade-off between coverage and resolution. Standard Hi-C provides a global view but often lacks the sequencing depth required to resolve fine-scale loops at specific gene clusters. Conversely, 3C or 4C are limited to single viewpoints.

Our 5C Sequencing Service (Chromosome Conformation Capture Carbon Copy) offers the perfect middle ground. By combining 3C library generation with ligation-mediated PCR (LM-PCR), 5C selectively amplifies millions of unique interactions occurring within specific genomic regions. This "many-to-many" approach creates a high-density matrix of interactions for targeted loci (typically 100 kb to 10 Mb), providing ultra-high resolution without the prohibitive cost of deep whole-genome sequencing.

This service is designed for researchers who need to dissect complex regulatory hubs—such as Hox clusters, globin loci, or GWAS intervals—where identifying every enhancer-promoter contact is critical.

Service Snapshot

  • Scope: "Many-to-Many" mapping within defined genomic intervals.
  • Resolution: Capable of detecting interactions at <5 kb resolution.
  • Efficiency: Focuses sequencing power exclusively on regions of interest.
  • Application: Fine-mapping regulatory networks and structural variation.

Why Choose 5C Sequencing?

Unmatched Resolution for Regulatory Hubs

Detecting specific loops between promoters and distal enhancers often requires immense sequencing depth in standard Hi-C. 5C achieves this depth efficiently by amplifying only the target region. This allows you to resolve complex "sub-TAD" structures and regulatory cliques that are often invisible or appear as noise in global maps.

Cost-Effective Deep Sequencing

By "copying" and sequencing only the interactions involving your target loci (e.g., 1% of the genome), 5C drastically reduces sequencing costs compared to achieving the same resolution with Hi-C. This maximizes the signal-to-noise ratio, ensuring that your reads contribute directly to the answer you need.

Customizable Primer Design

The success of a 5C experiment hinges on the design of the "Carbon Copy" primers. We utilize sophisticated algorithms to design alternating forward and reverse primers that anneal specifically to restriction fragments across your target region. This ensures comprehensive coverage and minimizes non-specific amplification.

Service Modules: Flexible Targeting Options

Targeted 5C Panels

Designed for mapping continuous genomic regions, such as a 2 Mb GWAS interval or a developmental gene cluster. This approach uses a tiled primer design to generate a complete, dense interaction matrix of the entire locus, revealing the full local topology.

Multiplex 5C

Ideal for mapping interactions between a dispersed set of genomic elements that are not necessarily contiguous. For example, if you want to map interactions between 50 specific promoters and 200 candidate enhancers scattered across different chromosomes, Multiplex 5C can selectively profile this "matrix of interest."

Technical Comparison: 3C vs. 4C vs. 5C vs. Hi-C

Feature 3C 4C Sequencing 5C Sequencing Hi-C Sequencing
Scope One-vs-One One-vs-All Many-vs-Many All-vs-All
Throughput Low (Single PCR) Medium (Sequencing) High (Multiplex Seq) Very High (Genome-wide)
Targeting Specific Locus Specific Viewpoint Specific Region(s) Whole Genome
Resolution High High High (<5 kb) Variable (Depth dependent)
Primary Use Validation Discovery from 1 site Network mapping in a region Global Architecture

Our Workflow: From 3C Library to Carbon Copy

Our streamlined 5C workflow transforms a standard chromatin conformation capture library into a focused sequencing pool.

  1. 3C Library Generation: Cells are crosslinked with formaldehyde to freeze chromatin interactions. Chromatin is digested with a restriction enzyme (e.g., HindIII) and ligated under dilute conditions to form 3C templates.
  2. Primer Design & Synthesis: Custom 5C primers are designed to flank the ligation junctions of the target fragments. Forward and Reverse primers are designed to anneal to alternating restriction fragments across the region.
  3. Multiplex Annealing & Ligation: The 5C primer pool is annealed to the 3C library. A thermostable ligase joins the specific Forward and Reverse primers only if they are annealed to ligated 3C fragments (representing an interaction).
  4. LM-PCR & Library Prep: The ligated primer pairs (the "Carbon Copies" of the interactions) are amplified via PCR using universal tails. This creates a library enriched purely for the targeted interactions.
  5. NGS & Interaction Calling: The library is sequenced, and bioinformatics pipelines map the paired primers to the genome to quantify interaction frequencies.

Step-by-step workflow of 5C sequencing from 3C library generation to NGS.

Demo Results: Dense Local Matrices

5C data provides a level of clarity that is often striking compared to global methods.

  • High-Definition Heatmaps: While a standard Hi-C map of a 1 Mb region might appear pixelated or sparse, a 5C heatmap of the same region is dense and sharp. You can clearly distinguish individual loops, TAD boundaries, and even sub-TAD structures.
  • Virtual 4C Plots: From the comprehensive 5C dataset, we can extract "virtual" 4C tracks for any viewpoint within the region, allowing you to visualize the interaction profile of any specific promoter or enhancer.

High-resolution 5C heatmap compared to standard Hi-C data.

Case Study: Dissecting Chromatin Networks in the EDC Locus

The Epidermal Differentiation Complex (EDC) is a large genomic cluster containing many genes regulated during skin development. Understanding how these genes are coordinated requires mapping the physical interactions across the entire locus. A 2017 study used 5C Sequencing to unravel this complex network.

The researchers designed a 5C panel covering the 3 Mb EDC locus. They performed the assay in skin epithelial cells to identify chromatin loops associated with gene expression changes during differentiation.

The 5C analysis revealed that the EDC locus is organized into distinct chromatin interaction networks. Interestingly, the method distinguished between "gene-rich" and "gene-poor" Topologically Associating Domains (TADs). In the gene-rich regions, 5C identified a dense network of specific promoter-enhancer loops that correlated with the activation of differentiation genes. These fine-scale interactions were crucial for understanding the coordinated regulation of the gene cluster.

PLOS Genetics figure illustrating 5C looping interactions across a locus and their association with promoter and enhancer regions.

5C sequencing provided the resolution needed to dissect the internal architecture of a specific gene cluster, revealing regulatory logic that would have been obscured in lower-resolution global maps.

(Source: Poterlowicz K. et al. 5C analysis of the Epidermal Differentiation Complex locus reveals distinct chromatin interaction networks between gene-rich and gene-poor TADs in skin epithelial cells. PLOS Genetics, 2017.)

Frequently Asked Questions

References

  1. Chromosome Conformation Capture Carbon Copy (5C): A massively parallel solution for mapping interactions between genomic elements. Genome Research. 2006.
  2. 5C analysis of the Epidermal Differentiation Complex locus reveals distinct chromatin interaction networks between gene-rich and gene-poor TADs in skin epithelial cells. PLOS Genetics. 2017.
  3. 3DeFDR: statistical methods for identifying cell type-specific looping interactions in 5C and Hi-C data. Genome Biology. 2020.
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