Introduction

Understanding the three-dimensional organization of the genome is essential for studying gene regulation, chromosome architecture, and genome evolution. While traditional Hi-C has become a widely used technique for chromatin conformation analysis, it primarily captures pairwise DNA interactions, making it difficult to resolve higher-order chromatin structures and complex genomic regions.

As genome research advances toward telomere-to-telomere (T2T) assemblies, chromosome-scale scaffolding, and analysis of polyploid or highly repetitive genomes, more powerful technologies are required. Nanopore Pore-C Sequencing addresses these challenges by combining chromatin conformation capture with long-read Oxford Nanopore sequencing, enabling direct characterization of multi-way chromatin interactions and native DNA methylation within a single experiment.

This integrated approach provides deeper insights into genome organization while improving chromosome assembly, haplotype phasing, and epigenetic analysis, making it an ideal solution for researchers in genomics, agriculture, biotechnology, and biomedical sciences.

What is Pore-C Sequencing?

Pore-C Sequencing is an advanced long-read chromatin conformation capture technology that extends the capabilities of conventional Hi-C. By integrating proximity ligation with Oxford Nanopore sequencing, Pore-C generates ultra-long reads containing multiple interacting genomic fragments, allowing researchers to investigate complex chromatin interactions at unprecedented resolution.

Unlike Hi-C, which predominantly detects pairwise contacts, Pore-C captures multi-way chromatin interactions directly from individual DNA molecules while simultaneously preserving native DNA methylation information.

How Pore-C Sequencing Works

1. Chromatin Crosslinking & Digestion

Chromatin is chemically crosslinked to preserve native DNA interactions, followed by restriction enzyme digestion to generate DNA fragments.

2. Proximity Ligation

DNA fragments that are physically close within the nucleus are ligated together, creating long concatemer molecules that retain three-dimensional chromatin interaction information.

3. Nanopore Long-Read Sequencing

The ligated concatemers are sequenced using Oxford Nanopore technology, producing ultra-long reads that contain multiple interacting genomic loci within a single sequencing read.

4. Integrated Genomic & Epigenetic Analysis

Because native DNA molecules are sequenced directly, Pore-C simultaneously captures:

• • Multi-way chromatin interactions
• • Chromosome-scale structural organization
• • Native DNA methylation and selected base modifications
• • Long-range genomic connectivity for improved genome assembly

Why Choose Pore-C?

Compared with conventional chromatin conformation capture methods, Pore-C provides several unique advantages:

• • Direct detection of multi-way chromatin interactions rather than only pairwise contacts.

• • Long-read sequencing for improved chromosome scaffolding and telomere-to-telomere genome assembly.
• • Simultaneous analysis of chromatin architecture and DNA methylation within a single workflow.
• • Enhanced haplotype phasing and resolution of repetitive or polyploid genomes.
• • Comprehensive insights into genome organization, structural variation, and epigenetic regulation using one integrated sequencing platform.
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Why Pore-C Sequencing Matters

Pore-C Sequencing provides a significant advancement over conventional chromatin conformation capture technologies by revealing the true complexity of three-dimensional genome organization. Instead of measuring only pairwise DNA interactions, Pore-C captures multiple interacting genomic regions within a single long read, offering a more comprehensive view of chromosome architecture.

This additional long-range information substantially improves chromosome-scale genome assembly, enhances telomere-to-telomere (T2T) genome construction, resolves highly repetitive regions such as centromeres, and increases scaffolding accuracy for complex and polyploid genomes.

Another key advantage is the ability to simultaneously detect native DNA methylation, enabling researchers to investigate genome structure and epigenetic regulation within the same sequencing experiment.

Pore-C vs. Hi-C

Advantages of Pore-C Over Hi-C

• Capture Higher-Order Chromatin Architecture

Identify multiple chromatin interactions simultaneously to better understand chromosome folding, long-range regulatory interactions, and genome organization.

• Improve Genome Assembly Quality

Long-read interaction data enhance chromosome scaffolding, reduce assembly ambiguities, and support high-quality telomere-to-telomere (T2T) genome assemblies.

• Resolve Complex Genomic Regions

Accurately characterize centromeres, telomeres, repetitive elements, and structurally complex genomic regions that are challenging for short-read technologies.

• Integrated Structural and Epigenetic Insights

Analyze chromatin organization and native DNA methylation together, providing a more comprehensive understanding of genome regulation.

• Better Performance for Polyploid Genomes

Long-read chromatin interaction data improve haplotype phasing, reduce false scaffold joins, and increase assembly accuracy for polyploid plants and other genetically complex species.

• Efficient Genome Scaffolding

The rich long-range interaction information generated by Pore-C can provide highly effective chromosome anchoring while reducing the sequencing depth typically required by conventional short-read chromatin conformation capture methods.

Service Workflow

Applications of Pore-C Sequencing

Pore-C Sequencing combines long-read chromatin conformation capture with Oxford Nanopore technology to deliver comprehensive insights into three-dimensional genome organization, chromosome-scale assembly, and epigenetic regulation. By simultaneously capturing multi-way chromatin interactions and native DNA methylation, Pore-C enables a wide range of applications across genomics, agriculture, biotechnology, and biomedical research.

Chromosome-Scale Genome Assembly

Pore-C provides long-range interaction data that improves genome scaffolding and chromosome assembly.

• •  Strengthens contig anchoring for chromosome-scale and telomere-to-telomere (T2T) genome assemblies.
• • Improves resolution of centromeres, telomeres, and highly repetitive genomic regions.
• • Enhances scaffold accuracy while reducing assembly ambiguities.
• • Supports structural variant discovery in complex genomes.

Polyploid Genome Analysis

Long-read chromatin interaction data help resolve genetically complex genomes.

• • Improves separation of homologous chromosomes and subgenomes.
• • Reduces scaffold misjoins in polyploid species.
• • Supports accurate haplotype phasing and comparative genomics.
• • Facilitates evolutionary and population genetics studies.

Three-Dimensional Genome Organization

Pore-C captures higher-order chromatin interactions that cannot be fully resolved using conventional Hi-C.

• • Maps multi-way chromatin interaction networks.
• • Investigates chromosome folding and nuclear architecture.
• • Studies long-range gene regulatory interactions.
• • Explores chromatin organization during development and disease.

Epigenetics & Chromatin Biology

Native Nanopore sequencing enables simultaneous structural and epigenetic analysis.

• • Detects DNA methylation together with chromatin interactions.
• • Investigates epigenetic regulation within three-dimensional genome architecture.
• • Supports studies of gene expression, genomic imprinting, chromatin accessibility, and transcriptional regulation.

Plant & Animal Genomics

Pore-C is well suited for agricultural and livestock genomics research.

• • Supports pan-genome construction and comparative genomics.
• • Identifies structural genomic features associated with agronomic and disease-resistance traits.
• • Accelerates molecular breeding and crop improvement programs.
• • Enables high-quality reference genome development for economically important species.

Recommended Sequencing Strategies

The optimal sequencing strategy depends on the desired assembly quality and genome complexity. Combining Pore-C with complementary sequencing technologies provides the best results for chromosome-scale and telomere-to-telomere genome assemblies.

Actual sequencing depth may vary depending on genome size, complexity, ploidy, and project objectives.

Bioinformatics Analysis

At N2Jenomics Lab Pvt. Ltd., our bioinformatics pipeline is specifically optimized for Nanopore Pore-C Sequencing, enabling accurate extraction of long-range chromatin interactions for genome assembly and 3D genome analysis.

• Optimized Analysis Workflow

Unlike conventional Hi-C pipelines, which typically fragment reads before alignment, our workflow is designed specifically for long-read Pore-C data.

• Full-Length Read Alignment

Entire Nanopore reads are aligned to the reference genome to preserve long-range chromatin information.

• Fragment Identification

Restriction fragments are identified after alignment, reducing mapping ambiguity and improving interaction accuracy.

• Multi-Way Contact Extraction

Chromatin contacts involving multiple genomic loci are extracted to generate higher-order interaction networks.

• Chromosome Scaffolding & Assembly Support

Long-range interaction data improve chromosome anchoring, scaffold ordering, and assembly validation.

• DNA Methylation Profiling

Native DNA methylation is analyzed directly from Nanopore sequencing data, providing integrated structural and epigenetic information.

• Visualization & Reporting

Generate publication-ready contact heatmaps, interaction networks, methylation tracks, and comprehensive project reports.

Quality Control Metrics

Our bioinformatics workflow includes specialized quality assessment metrics for evaluating Pore-C datasets.

• • Mean fragment count per read
• • Contacts-per-read ratio
• • Valid interaction percentage
• • Average interaction distance
• • Cis/trans interaction ratio
• • Read length distribution
• • Contact map quality assessment
• • Sequencing yield and alignment statistics

• These metrics help ensure robust data quality and reliable downstream biological interpretation.

Deliverables

Each Pore-C project includes a comprehensive set of sequencing data and bioinformatics outputs.

• • Raw sequencing data (FASTQ)
• • Alignment files (BAM) (optional)
• • Multi-way chromatin interaction datasets
• • Chromatin contact matrices and heatmaps
• • Three-dimensional interaction network visualizations
• • DNA methylation analysis (optional)
• • Chromosome scaffolding results (optional)
• • Quality control reports with sequencing and interaction statistics
• • Publication-ready figures and a comprehensive bioinformatics report
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Pore-C Data Quality Assurance

At N2Jenomics Lab Pvt. Ltd., we implement stringent quality control measures throughout the Pore-C workflow to ensure reliable chromatin interaction data and high sequencing performance. From sample assessment to final data delivery, every stage is optimized to maximize data quality and reproducibility.

Sequencing Performance

Our Pore-C sequencing workflow is designed to generate high-quality long-read datasets suitable for chromosome-scale genome assembly and three-dimensional genome analysis.

Actual sequencing performance depends on sample quality, genome complexity, library preparation, and sequencing depth.

Recommended Sample Types

For optimal sequencing performance, we recommend submitting high-quality biological material.

• Plant Samples

• - Young, actively growing leaves are preferred.
• - Minimize contamination from polysaccharides and secondary metabolites whenever possible.

• Animal Samples

• - Fresh blood is recommended when appropriate.
• - Fresh or snap-frozen tissues, such as liver and muscle, generally provide excellent results.

• Challenging Sample Types

Some samples may require customized extraction and library preparation workflows, including:

• - Plant species rich in secondary metabolites
• - Marine organisms and aquatic plants
• - Algae
• - Insects
• - Amphibians
• - Birds
• - Rabbits and other difficult tissue types

Our technical team will evaluate sample suitability and recommend the most appropriate workflow before project initiation.

Sample Requirements

To obtain the highest-quality chromatin interaction data, samples should be properly collected, preserved, and transported.

Storage & Shipping

• •  Snap-freeze samples immediately in liquid nitrogen whenever possible.
• • Store samples at −80°C before shipment.
• • Ship samples on dry ice to maintain sample integrity.
• • Avoid repeated freeze–thaw cycles.
• • Ensure all sample tubes are tightly sealed to prevent leakage during transport.

Sample Labeling

• • Clearly label every tube with a unique sample ID.
• • Ensure sample identifiers match the submitted Sample Information Form.
• • Unless otherwise advised, avoid sending substantially more material than the recommended input amount.

Why Choose N2Jenomics Lab Pvt. Ltd.?

N2Jenomics Lab Pvt. Ltd. provides a complete, end-to-end Nanopore Pore-C Sequencing solution, combining advanced laboratory workflows with expert bioinformatics support to deliver high-quality chromosome conformation data for complex genome research.

• Experienced Scientific Team

Our genomics specialists have extensive expertise in chromatin conformation capture technologies, long-read sequencing, genome assembly, and epigenetic analysis.

• Optimized Experimental Workflows

Every stage—from chromatin crosslinking and library preparation to sequencing—is carefully optimized to maximize data quality, library complexity, and reproducibility.

• High-Throughput Oxford Nanopore Sequencing

Access to state-of-the-art PromethION sequencing platforms enables efficient processing of projects ranging from pilot studies to large-scale genome initiatives.

• Advanced Bioinformatics Analysis

Our dedicated bioinformatics team provides comprehensive analysis, including chromosome scaffolding, multi-way chromatin interaction mapping, DNA methylation profiling, genome assembly support, visualization, and publication-ready reporting.

• Comprehensive Quality Control

Multiple quality assessment checkpoints are incorporated throughout the workflow to ensure reliable sequencing performance and robust downstream analyses.

• End-to-End Project Support

From experimental design and sample preparation guidance to data interpretation and technical consultation, our experts work closely with researchers to ensure successful project outcomes.

1. What is Pore-C Sequencing, and how does it differ from Hi-C?

Pore-C Sequencing is a long-read chromatin conformation capture technology that combines proximity ligation with Oxford Nanopore sequencing. Unlike conventional Hi-C, which primarily measures pairwise chromatin interactions, Pore-C captures multiple interacting genomic loci within a single sequencing read. It also preserves native DNA methylation, enabling simultaneous analysis of genome architecture and epigenetic modifications while improving chromosome scaffolding and assembly accuracy.

2. Can Pore-C detect DNA methylation and chromatin interactions in the same experiment?

Yes. Because Pore-C uses native Oxford Nanopore sequencing without PCR amplification, it retains DNA base modification information. This allows researchers to analyze three-dimensional chromatin interactions and native DNA methylation from the same dataset, providing integrated structural and epigenetic insights.

3. What sample types are suitable for Pore-C Sequencing?

Pore-C is compatible with a wide variety of biological samples, including cultured cells, plant tissues, animal tissues, microbial samples, and fresh or properly preserved biological materials. Samples should be appropriately crosslinked to preserve chromatin interactions prior to library preparation. Our technical team can provide detailed sample preparation and fixation guidelines based on your project.

4. What data and deliverables will I receive?

Depending on the selected analysis package, project deliverables may include:

• • Raw sequencing data (FASTQ)

• • Alignment files (BAM)
• • Quality control reports
• • Chromatin contact matrices and interaction heatmaps
• • Multi-way chromatin interaction networks
• • DNA methylation analysis
• • Chromosome scaffolding results (optional)
• • Comprehensive bioinformatics report with publication-ready figures

5. How much sequencing depth is recommended for Pore-C projects?

The required sequencing depth depends on genome size, genome complexity, assembly objectives, and downstream analyses. Chromosome-scale scaffolding generally requires moderate coverage, whereas telomere-to-telomere (T2T) assemblies, polyploid genomes, and highly repetitive genomes typically benefit from deeper sequencing. Our scientists will recommend an optimal sequencing strategy based on your research goals.

6. Which bioinformatics analyses are included?

Our standard and optional bioinformatics workflows include:

• • Basecalling and quality assessment
• • Read alignment and contact extraction
• • Multi-way chromatin interaction analysis
• • Contact matrix and heatmap generation
• • Chromosome scaffolding support
• • DNA methylation profiling
• • Genome assembly integration
• • Data visualization and comprehensive project reporting

7. What research applications is Pore-C best suited for?

Pore-C Sequencing is ideal for chromosome-scale genome assembly, telomere-to-telomere (T2T) genome projects, three-dimensional genome organization studies, polyploid genome analysis, haplotype phasing, epigenetic research, structural genomics, and comparative genomics.

8. Why choose N2Jenomics Lab for Pore-C Sequencing?

N2Jenomics Lab Pvt. Ltd. provides a complete end-to-end Pore-C sequencing solution, including experimental design, sample quality assessment, library preparation, Oxford Nanopore sequencing, advanced bioinformatics analysis, chromosome scaffolding, and publication-ready reporting. Our experienced genomics and bioinformatics team delivers reliable, high-quality data tailored to the requirements of both academic and industrial research projects.