HiFi-C Sequencing Service | Ultra-Accurate 3D Genome Reconstruction with PacBio HiFi

At CD Genomics, we provide advanced HiFi-C sequencing (CiFi sequencing) services that combine chromosome conformation capture (3C) with PacBio HiFi long-read sequencing.

This integrated approach delivers high-fidelity, multi-contact chromatin interaction maps across the entire genome—enabling precise 3D structural reconstruction even in complex, repetitive, or polyploid species.

Designed for research, drug discovery, and crop improvement, our HiFi-C service overcomes the limitations of short-read Hi-C and noisy long-read Pore-C.

With exceptional read accuracy (Q40, 99.99%), minimal input requirements (as few as 60,000 cells), and end-to-end analytical support, CD Genomics empowers researchers to explore genome organization with unmatched clarity and efficiency.

We help clients overcome challenges such as:

• Poor resolution of Hi-C in repetitive and centromeric regions
• High material demand in traditional long-read 3C sequencing
• Difficulty scaffolding polyploid and highly repetitive genomes

Why choose CD Genomics HiFi-C?

• PacBio HiFi Q40 accuracy ensures reliable chromatin contact detection
• Works efficiently with low-input and rare samples
• Captures multiple interaction sites in a single molecule
• Ideal for T2T genome assembly, structural variant mapping, and epigenomic regulation studies

At a glance:

• What Is HiFi-C Sequencing?
• Why Choose HiFi-C over Hi-C or Pore-C?
• Key Applications of HiFi-C Sequencing
• Workflow Overview
• Recommended Sequencing Depth for HiFi-C
• HiFi-C Bioinformatics Analysis Overview
• HiFi-C Sequencing Sample Requirements
• Case Study – Validation of HiFi-C Technology through the CiFi Method
• Frequently Asked Questions about HiFi-C Sequencing

What Is HiFi-C Sequencing?

HiFi-C sequencing—also known as CiFi sequencing —is a next-generation chromosome conformation capture (3C) technology built on PacBio HiFi long-read sequencing. It provides base-level accuracy for mapping the three-dimensional genome, revealing how chromatin folds, interacts, and regulates gene expression.

Traditional Hi-C, based on short-read sequencing, captures only pairwise DNA interactions, limiting its resolution in repetitive or structurally complex regions.

By contrast, HiFi-C directly sequences multi-contact DNA fragments, allowing a single long read to represent several interacting loci within the same molecule. This enables precise 3D genome reconstruction with fewer assumptions and reduced signal noise.

HiFi-C retains the principles of the 3C technique—crosslinking, digestion, ligation, and sequencing—but pairs them with HiFi reads that combine long read length (5–10 kb) and Q40-level accuracy (99.99%).

As a result, researchers can resolve centromeres, telomeres, and segmental duplications that are usually inaccessible to Hi-C or Pore-C.

Key advantages of the HiFi-C platform include:

• Single-molecule multi-contact detection for higher structural completeness
• Exceptional mapping precision in repetitive and low-complexity regions
• Low input requirement suitable for rare or precious samples
• Compatibility with both model and non-model species

These attributes make HiFi-C an essential tool for genome assembly validation, structural variant detection, and 3D epigenetic research—a major step forward in spatial genomics.

Why Choose HiFi-C over Hi-C or Pore-C?

Researchers increasingly need 3D genome data that are both accurate and biologically complete. HiFi-C sequencing achieves this balance better than any other chromatin conformation technology by combining the precision of PacBio HiFi reads with the spatial mapping power of 3C-based chemistry.

A. Proven Technical Superiority

HiFi-C delivers long, high-fidelity reads with Q40 (99.99%) accuracy, allowing exact identification of restriction sites and chromatin contacts.

This eliminates the mapping ambiguity common in short-read Hi-C and significantly improves the continuity of interaction signals across centromeric and repetitive regions.

• Higher valid data rate: 77–94%, far exceeding Pore-C's 38–76%
• Lower input requirement: as few as 60,000 cells per sample
• More usable data: each read contains multiple contacts, enabling direct reconstruction of long-range and high-order chromatin structures
• Optimized for precision: HiFi-C minimizes false contacts and sequencing noise, providing more confident structural inferences

B. Demonstrated Maturity and Real-World Validation

Unlike emerging methods still in optimization, HiFi-C has been validated across more than 200 species—including mammals, plants, and polyploid crops.

In direct comparisons, a 12.5× HiFi-C dataset produced stronger, continuous centromeric signals than a 120× Hi-C dataset.

Studies show that HiFi-C improves scaffold completeness and reduces mis-joins in genome assemblies, particularly for polyploid or repeat-rich species.

C. Comparative Summary

D. Summary Insight

HiFi-C combines the accuracy of short reads, the contact depth of long reads, and the scalability of modern 3C pipelines.

It is the most balanced solution for projects requiring precise 3D genome reconstruction, haplotype phasing, or T2T assembly validation—all with lower input and higher yield.

Recommended Reading:

Explore related 3D genome technologies such as ChIA-PET Sequencing and HiChIP Sequencing for protein-directed interaction profiling.

Key Applications of HiFi-C Sequencing

HiFi-C sequencing enables researchers to move beyond pairwise DNA contacts toward a full understanding of chromatin topology.

Its ability to capture multi-contact, high-fidelity reads makes it ideal for a broad range of biological and applied research applications—from structural genomics to functional regulation.

1. Chromosome-Level Genome Scaffolding and T2T Assembly

HiFi-C provides continuous long-range interaction data that connect contigs into chromosome-scale scaffolds.

By bridging repetitive or low-complexity regions, it supports telomere-to-telomere (T2T) assemblies with higher completeness and fewer gaps.

This makes HiFi-C particularly valuable for de novo genome assembly validation in both model and non-model organisms.

2. Structural Variant (SV) and Polyploid Genome Analysis

HiFi-C long reads can span large insertions, deletions, and inversions, allowing direct detection of how structural variants reshape 3D chromatin folding.

In polyploid genomes, HiFi-C clarifies haplotype-specific interactions and reduces scaffolding artefacts, offering clearer insight into allele-specific regulation and chromosomal organisation.

3. Repetitive and Heterochromatic Region Resolution

Traditional Hi-C struggles to map repetitive regions such as centromeres and telomeres.

HiFi-C's high-accuracy, long-read coverage ensures continuous contact signals across these challenging areas, providing unprecedented access to the structural cores of chromosomes.

4. 3D Regulatory Architecture Mapping

HiFi-C identifies topologically associated domains (TADs), enhancer–promoter loops, and other long-range interactions that underlie gene expression control.

When integrated with RNA-seq or ChIP-seq, it connects chromatin structure with transcriptional function, supporting mechanistic studies in developmental biology and disease models.

5. Comparative and Evolutionary 3D Genomics

Because of its reproducibility and cross-species compatibility, HiFi-C is used to compare genome folding across species, developmental stages, or environmental conditions—revealing how chromatin architecture evolves to support diverse biological functions.

6. Multi-Omics Integration

HiFi-C integrates seamlessly with ATAC-seq, RNA-seq, and ChIP-seq, allowing construction of multi-dimensional regulatory maps that connect genome organisation, chromatin accessibility, and transcriptional output.

Workflow Overview

CD Genomics provides a comprehensive HiFi-C sequencing workflow that integrates sample preparation, PacBio HiFi sequencing, and data interpretation into a single, quality-controlled pipeline.

Each stage is optimized to preserve chromatin integrity, maximize contact recovery, and deliver reproducible 3D genome insights.

Step 1. Chromatin Crosslinking and Digestion

Cells or tissues are fixed to capture native chromatin interactions, then digested with restriction enzymes such as DpnII or HindIII.

This process preserves both short- and long-range DNA contacts essential for downstream structural mapping.

Step 2. Chromatin Interaction Linking and DNA Purification

Crosslinked fragments are ligated to create long concatemeric DNA molecules that represent multi-contact events.

Purified ligation products retain the physical structure of chromatin within the nucleus, forming the foundation of the HiFi-C library.

Step 3. PacBio HiFi Library Preparation

Purified 3C products are converted into SMRTbell templates suitable for PacBio HiFi sequencing.

The long reads generated maintain the original ligation structure and achieve Q40-level accuracy for precise contact site identification.

Step 4. HiFi Sequencing on the PacBio Revio Platform

Sequencing is conducted using PacBio Revio, which provides exceptional yield and read accuracy.

The platform supports combined HiFi whole-genome and HiFi-C sequencing runs, reducing time and experimental variability.

Step 5. Data Delivery and Reporting

Raw and processed data are provided alongside quality metrics, including valid read percentage, contact depth, and cis/trans interaction ratios.

A full interpretive report summarises chromatin architecture, assembly improvement, and regulatory insights.

Recommended Reading:

For a deeper understanding of long-read 3C methods, see our Chromatin Conformation Sequencing Overview.

Recommended Sequencing Depth for HiFi-C

To help planning and budgeting, CD Genomics recommends the following HiFi-C sequencing depths by research goal. Values are per sample unless noted.

Notes

• Depth refers to HiFi-C coverage on the target genome; adjust for genome size and sample complexity.
• For low-input or repeat-rich species, consider the higher end of each range.
• We offer custom designs for rare samples and multi-omics integration (HiFi-C + RNA-seq/ATAC-seq).

HiFi-C Bioinformatics Analysis Overview

HiFi-C Sequencing Sample Requirements

Case Study – Validation of HiFi-C Technology through the CiFi Method

Source: McGinty S.P. et al. (2025) CiFi: Accurate long-read chromatin conformation capture with low-input requirements. bioRxiv https://doi.org/10.1101/2025.01.31.635566

1. Background

The CiFi study was designed to test whether combining chromosome conformation capture (3C) with PacBio HiFi long-read sequencing could overcome the limitations of short-read Hi-C.

Hi-C lacks the ability to resolve complex or repetitive regions such as centromeres and telomeres, while long-read 3C methods like Pore-C require millions of cells.

The authors sought to create a method capable of accurate multi-contact detection with minimal input DNA, setting the foundation for what is now known as HiFi-C sequencing.

2. Methods

Researchers crosslinked chromatin, digested it with restriction enzymes (DpnII or HindIII), and performed 3C ligation to generate long concatemeric fragments.

These molecules were sequenced using PacBio HiFi technology on human GM12878 lymphoblastoid cells and two low-input animal models (the mosquito Anopheles and the Mediterranean fruit fly Ceratitis capitata).

Custom bioinformatics pipelines were applied to extract multi-contact reads, map them to reference genomes, and compute contact matrices spanning kilobase to chromosome-wide distances.

3. Results

• Higher accuracy in complex regions: In segmental duplications and centromeres, 83–89 % of HiFi-C reads achieved high mapping quality compared with 33–37 % for Illumina Hi-C.
• Low-input performance: Usable data were obtained from only ~62,000 cells (≈ 370 ng DNA), reducing input needs by >100× relative to Pore-C.
• Comprehensive chromatin coverage: Each read spanned a median of 17 contact sites (~350 bp per contact), revealing long-range interactions extending >100 Mb.
• Cross-species robustness: The same workflow assembled a ~600 Mb diploid fruit-fly genome at chromosome scale, proving adaptability to non-model organisms.

Comparison of chromatin contact maps generated by Illumina Hi-C and PacBio HiFi-C in human GM12878 cells. HiFi-C shows enhanced signal continuity and resolution across centromeric and repetitive regions.

4. Conclusions

CiFi (HiFi-C) delivers long-read precision and high valid-data yield at low input, providing a practical solution for 3D genome mapping across species.

The method excels in resolving repetitive, heterochromatic, and structurally complex regions, establishing HiFi-C as the preferred platform for:

• Chromosome-scale (T2T) assembly validation
• Structural-variant and haplotype-specific 3D analysis
• Small-sample or rare-cell epigenomic studies

These findings validate CD Genomics' adoption of the HiFi-C workflow as a reproducible, low-input, high-fidelity service for research, drug discovery, and agricultural genomics.

Frequently Asked Questions about HiFi-C Sequencing

Pore-C Sequencing Service

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HiChIP Sequencing Service

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Chromatin Conformation Sequencing