Sample Submission Guidelines
What Is CiFi Sequencing
CiFi (Chromosome conformation capture with HiFi sequencing) is a method that integrates chromosome conformation capture (3C) chemistry with PacBio HiFi long-read sequencing. Published in Nature Communications in December 2025 (McGinty et al.), CiFi was developed to overcome the two core limitations of conventional Hi-C sequencing: high cell-input requirements and poor mappability in repetitive genomic regions.
Standard short-read Hi-C requires millions of cells and produces paired 150 bp reads. When those reads fall within centromeres, segmental duplications, or other repeat-rich regions, they cannot be uniquely mapped — leaving the most biologically significant parts of the genome invisible to contact analysis. CiFi addresses both problems simultaneously. By applying whole-genome amplification to 3C libraries and sequencing the resulting long concatemers on PacBio HiFi, CiFi generates multi-kilobase reads (5–25 kb) that each carry multiple interacting genomic segments (350 bp to 2 kb per segment). These longer, multi-contact reads map uniquely across regions that defeat short reads, and the library preparation is sensitive enough to work from sub-microgram DNA inputs — as few as 60,000 cells.
The resulting data are largely concordant with conventional Hi-C for standard interaction detection, but unlock a series of additional capabilities: TAD boundary calling across centromeres, enhanced haplotype phasing, and chromosome-scale genome assembly scaffolding — all from a single sequencing run, optionally combined with HiFi whole-genome sequencing on the same instrument.
CiFi vs. Standard Hi-C: Where Long Reads Win
Both methods capture proximity-ligation chromatin contacts genome-wide. CiFi adds long-read accuracy, multi-contact information, and low-input sensitivity that short-read Hi-C cannot deliver.
| Feature | Standard Hi-C (Short-Read) | CiFi (PacBio HiFi Long-Read) |
|---|---|---|
| Minimum cell input | Typically ≥1 million cells | ≥60,000 cells (sub-microgram DNA) |
| Read length | 150 bp paired-end | 5–25 kb multi-contact HiFi reads |
| Contacts per read | 2 segments per read pair | Multiple segments per read (multi-contact) |
| Repetitive region mappability | Poor — centromeres, SDs, low-complexity regions missed | Improved — longer segments span repeats uniquely |
| Haplotype phasing | Limited — short reads rarely span heterozygous SNPs | Substantially enhanced — HiFi accuracy across phased blocks |
| TAD analysis in complex regions | Fails at centromeres and segmental duplications | Resolves TADs across disease-associated genomic hotspots |
| Genome assembly scaffolding | Compatible with Hi-C scaffolders | Competitive or fewer contacts needed vs. standard Hi-C |
| Co-sequencing with WGS | Requires separate library and sequencing run | Single SMRT Cell can produce WGS + CiFi library simultaneously |
| Small organism / single individual | Not feasible without pooling | Single small insect validated (Anopheles mosquito, fruit fly) |
Core Technical Capabilities
CiFi combines PacBio HiFi accuracy with 3C multi-contact library chemistry to deliver four integrated capabilities from a single experiment.
Ultra-Low Input Chromatin Profiling
- Starting material: ≥60,000 cells or sub-microgram genomic DNA
- Whole-genome amplification (WGA) of 3C libraries preserves contact information while amplifying limited input
- Validated on single insects (Anopheles coluzzii mosquito; Ceratitis capitata Mediterranean fruit fly)
- Opens chromatin 3D profiling to previously inaccessible sample types: rare cell populations, precious biopsies, individual small organisms
Repetitive Region Resolution
- Segments of 350 bp to 2 kb per concatemer enable unique mapping in centromeres and segmental duplications
- Gains in TAD/domain boundary calling across human disease-associated genomic hotspots
- Improved coverage uniformity across satellite repeat arrays, tandem duplications, and low-complexity loci
- Pairwise interactions concordant with conventional Hi-C for euchromatic regions, with additional resolution in heterochromatin
Haplotype-Resolved Phasing
- HiFi base accuracy (>99%) across phased genomic segments enables confident heterozygous SNP calling within contact reads
- Multi-contact reads provide long-range phase blocks substantially improved over short-read Hi-C
- Phase block extension across complex structural variants and repeat boundaries
- Integrates with standard HiFi de novo genome assembly pipelines (Hifiasm, HiCanu)
Chromosome-Scale Assembly Scaffolding
- CiFi contacts scaffold HiFi contig assemblies to chromosome scale — same data pipeline as Hi-C scaffolding, improved by longer reads
- Demonstrated: chromosome-scale phased diploid assembly of a single Mediterranean fruit fly from one SMRT Cell
- Compatible or lower contact requirements vs. traditional Hi-C for scaffolding equivalent genome sizes
- Enables single-individual genome projects for organisms with limited material
Service Workflow
From sample submission to publication-ready chromatin interaction data and genome assembly outputs.
Step 1 — Consultation & Sample QC: We review your genome size, organism, available cell number, and project goals (3D chromatin analysis only, or combined WGS+CiFi single-run assembly). All samples undergo cell count verification and nucleic acid QC before processing. Detailed sample handling guidance is provided upon project initiation to preserve chromatin integrity during shipping.
Step 2 — 3C Library Preparation: Cells are crosslinked with formaldehyde to preserve chromatin contacts, digested with a frequent-cutting restriction enzyme (DpnII or HindIII depending on genome GC content), and proximity-ligated under dilute conditions that favor in situ junctions reflecting real spatial proximity. Long ligated fragments are purified and size-selected to maximize multi-kb concatemer yield.
Step 3 — WGA Amplification & PacBio HiFi Sequencing: Whole-genome amplification is applied to the 3C library to reach PacBio input requirements without losing contact information. PacBio-compatible SMRTbell adaptors are ligated and libraries are sequenced on PacBio Revio using circular consensus sequencing (CCS/HiFi) mode, generating per-read accuracy >99% across multi-kb concatemers. When combined with HiFi WGS, both libraries can be processed from the same individual.
Step 4 — Multi-Contact Bioinformatics Analysis: HiFi reads are processed to extract individual segments, assign V(D)J-equivalent contact pairs, and generate genome-wide interaction maps. TAD/compartment/loop calling is performed with established tools (juicer, HOMER, or equivalent). Haplotype phasing integrates CiFi multi-contact information with HiFi contig phase blocks. For assembly projects, CiFi contacts scaffold HiFi contigs to chromosome scale using 3D-DNA or YaHS.
Step 5 — Results Delivery: You receive raw HiFi FASTQ files, per-sample QC metrics, pairwise interaction matrices (.cool/.hic), chromatin domain annotation files, haplotype phasing output, and — for assembly projects — a chromosome-scale scaffolded FASTA with assembly statistics. A scientific consultation call is included to discuss results and downstream applications.
Key Applications
CiFi extends chromatin 3D profiling and genome assembly to sample types and genomic regions that have been inaccessible to conventional Hi-C.
Non-Model Organism Genome Assembly & Scaffolding
For organisms without reference genomes and with limited tissue availability — insects, parasites, endangered species, agricultural pests — CiFi provides the chromatin contact data needed to scaffold HiFi contig assemblies to chromosome scale. The low-input requirement eliminates the pooling of multiple individuals, preserving biological accuracy.
3D Chromatin Analysis in Repeat-Rich & Complex Regions
Centromeres, pericentromeric heterochromatin, segmental duplications, and disease-associated genomic hotspots are invisible to standard Hi-C because short reads cannot map uniquely there. CiFi's longer segments resolve TAD boundaries and loop structures in these regions, revealing chromatin organization that conventional methods miss. See our overview of Hi-C sequencing for background on chromatin domain biology.
Single-Individual Genome Projects
Conservation genomics, population genetics of rare taxa, and forensic entomology all require genome-scale data from a single organism. CiFi was validated on single Anopheles coluzzii mosquitoes (chromatin profiling) and a single Ceratitis capitata Mediterranean fruit fly (chromosome-scale phased diploid assembly). Both projects used only one SMRT Cell, demonstrating the cost efficiency of the approach for small-organism genomics.
Haplotype-Resolved Diploid Assembly
When combined with PacBio HiFi whole-genome sequencing, CiFi provides the long-range phase information needed to separate maternal and paternal haplotypes across entire chromosomes. This enables fully phased diploid assemblies from outbred individuals without trio sequencing — applicable to polyploid plant genomes, highly heterozygous animal genomes, and human disease research. Learn more about genome assembly approaches for complex organisms.
Rare & Precious Sample Chromatin Profiling
Tumor biopsies, sorted cell populations, micro-dissected tissue sections, and historical museum specimens contain too few cells for conventional Hi-C. CiFi's 60,000-cell minimum opens 3D genome organization studies to these sample types — without sacrificing interaction map quality for euchromatic regions.
Sample Requirements
Chromatin integrity is critical for 3C-based methods. All samples should be crosslinked or flash-frozen immediately upon collection. Contact our team before shipping for handling SOPs specific to your organism and sample type.
| Sample Type | Recommended Input | Minimum Input | Notes |
|---|---|---|---|
| Live cell suspension (mammalian) | 1–5 million cells | 60,000 cells | Process immediately; crosslink on-site or ship in media on ice |
| Crosslinked cell pellet | 1–5 million cell equivalents | 60,000 cell equivalents | Flash-frozen after crosslinking; ship on dry ice |
| Fresh tissue | 20–50 mg | 5 mg | Dissociate to single-cell suspension before crosslinking; or ship in crosslinking buffer |
| Single small insect / whole organism | 1 individual | 1 individual | Ship in ethanol (museum specimens) or flash-frozen; contact us for pre-QC protocol |
| Pre-crosslinked nucleus suspension | 500,000 nuclei | 60,000 nuclei | In FACS buffer; ship on dry ice |
| High-molecular-weight genomic DNA | ≥500 ng | Sub-microgram (contact us) | For WGA-amplified 3C only; contact team to confirm feasibility |
- Restriction enzyme selection: DpnII is recommended for most organisms; HindIII for higher coverage of GC-rich genomes. We advise on enzyme choice during pre-project consultation.
- Combined WGS+CiFi projects: Additional HMW gDNA is required for the WGS library component. Typical requirement: ≥2 µg total HMW gDNA from the same individual.
Bioinformatics Analysis & Deliverables
Our CiFi bioinformatics pipeline processes multi-contact HiFi reads through a validated analytical workflow covering all standard chromatin interaction outputs plus the long-read-specific enhancements unique to CiFi data.
- Raw Data & QC: Demultiplexed HiFi FASTQ files with per-sample QC metrics — CCS pass rate, per-read accuracy, subread coverage, library complexity.
- Segment Extraction & Mapping: Multi-contact concatemer reads are segmented, mapped to reference (or de novo assembly), and filtered for unique alignments. Cis/trans contact ratio and P(s) decay curves are reported.
- Pairwise Interaction Maps: Genome-wide contact matrices in .cool and .hic formats at multiple resolutions (10 kb, 25 kb, 100 kb), compatible with Juicebox, HiGlass, and cooltools visualization.
- Chromatin Domain Analysis: A/B compartment calls, TAD boundary identification, and chromatin loop calling — with specific reporting for complex regions (centromeres, segmental duplications) where CiFi outperforms standard Hi-C.
- Haplotype Phasing: Phase block extension using CiFi multi-contact reads integrated with HiFi assembly phase information. Phased VCF and haplotype-separated contact matrices delivered.
- Assembly Scaffolding (when applicable): Chromosome-scale FASTA from HiFi contig assembly scaffolded with CiFi contacts. Assembly statistics: N50, scaffold count, BUSCO completeness, chromosome-scale confirmation.
Integration with PacBio SMRT sequencing data from external sources or prior projects is available. Custom analyses — including multi-species comparisons, allele-specific interaction quantification, and epigenome data integration — are available upon request.
References
- McGinty SP, Kaya G, Sim SB, Makunin A, Corpuz RL, Quail MA, Abuelanin M, Lawniczak MKN, Geib SM, Korlach J, Dennis MY. CiFi: accurate long-read chromosome conformation capture with low-input requirements. Nat Commun. 2025;17:215. https://doi.org/10.1038/s41467-025-66918-y
- Lieberman-Aiden E, van Berkum NL, Williams L, et al. Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science. 2009;326(5950):289–293. https://doi.org/10.1126/science.1181369
- Rao SS, Huntley MH, Durand NC, et al. A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping. Cell. 2014;159(7):1665–1680. https://doi.org/10.1016/j.cell.2014.11.021
For Research Use Only. Not for use in diagnostic or clinical procedures.
Demo Results
Genome-wide CiFi contact matrix (human GM12878 lymphoblastoid cell line) showing improved interaction coverage across centromeric and segmental duplication regions (highlighted) compared to standard short-read Hi-C. (McGinty SP et al., Nat Commun, 2025)
Chromosome-scale phased diploid assembly of a single Mediterranean fruit fly (Ceratitis capitata) produced by combining HiFi whole-genome sequencing and CiFi contacts from one individual. CiFi scaffolded HiFi contigs into chromosome-scale sequences. (McGinty SP et al., Nat Commun, 2025)
References
- McGinty SP et al. CiFi: accurate long-read chromosome conformation capture with low-input requirements. Nat Commun. 2025;17:215. https://doi.org/10.1038/s41467-025-66918-y
CiFi Sequencing FAQs
1. How does CiFi differ from Pore-C or standard HiFi-C methods?
CiFi, Pore-C, and HiFi-C all combine long-read sequencing with 3C chemistry, but differ in key aspects. CiFi uses PacBio HiFi sequencing with whole-genome amplification of 3C libraries, achieving >99% per-read accuracy and enabling sub-microgram input. Pore-C uses Oxford Nanopore without amplification, requiring higher input and trading base-level accuracy for longer raw reads. HiFi-C is the broader term for PacBio-based 3C approaches, of which CiFi is a specific validated protocol with published performance benchmarks in Nature Communications. CD Genomics implements the CiFi protocol with the WGA amplification step that enables the 60,000-cell minimum input.
2. Can CiFi be used without a reference genome?
Yes. For organisms without a reference, CiFi contacts scaffold de novo HiFi contig assemblies to chromosome scale — the CiFi data effectively serves as the phasing and scaffolding layer on top of a HiFi-based draft assembly. This is the application demonstrated with the Ceratitis capitata Mediterranean fruit fly in the original CiFi publication. When sequencing a new organism, we recommend planning a combined WGS+CiFi project from the same individual to streamline data integration.
3. What organisms has CiFi been validated on?
The original CiFi paper validated the method on human lymphoblastoid cells (GM12878), a single Anopheles coluzzii mosquito (genome-wide chromatin interaction profiling), and a single Ceratitis capitata Mediterranean fruit fly (chromosome-scale phased diploid assembly). The method is organism-agnostic and applicable to any species where cells or tissue can be crosslinked for 3C library preparation. We have applied Hi-C and related long-read methods to a broad range of insects, plants, vertebrates, and fungi; contact us to discuss your specific organism.
4. Is CiFi compatible with FFPE or archived samples?
CiFi requires intact crosslinked chromatin for 3C library preparation, which means it requires fresh or flash-frozen tissue — not FFPE. Ethanol-preserved museum specimens (insects, small organisms) have been used successfully when handled with appropriate care. If your material is FFPE, we can advise on alternative chromatin capture strategies or alternative sequencing approaches for genomic analysis from archived material.
5. Can you perform CiFi from sorted cell populations or flow cytometry-isolated cells?
Yes. FACS-sorted cell populations are compatible with CiFi as long as sorting is performed under conditions that preserve cell membrane integrity for downstream crosslinking, and the recovered cell number meets the 60,000-cell minimum. We recommend coordinating the sorting protocol with our scientific team before processing to ensure chromatin integrity is maintained throughout.
CiFi Sequencing Case Studies
Customer Publication Highlight
Unraveling Forensic Timelines Using Molecular Markers in Phormia regina Maggots
Journal: PLOS Genetics
Impact Factor: 3.7
Published: 2025
Service Used: Hi-C Library Construction and Sequencing
Background
Estimating the post-mortem interval (PMI) in forensic investigations relies critically on understanding the development timeline of blow flies, particularly Phormia regina. Accurate molecular dating of developmental stages — from egg to pupal stage — requires access to high-quality genomic and transcriptomic reference data. However, generating chromosome-scale genome assemblies for insects is challenging due to the limited tissue available from individual specimens and the complexity of repetitive regions in dipteran genomes. This study employed Hi-C library construction and sequencing to provide the long-range contact data needed for chromosome-scale genome scaffolding, enabling subsequent transcriptomic profiling of developmental markers at each forensic-relevant life stage.
Materials & Methods
Sample Preparation
- Phormia regina blow fly specimens across developmental stages
- Fresh tissue collection for Hi-C crosslinking
- RNA extraction for transcriptome profiling at each developmental timepoint
Sequencing
- Hi-C library construction and sequencing (CD Genomics service)
- High-throughput chromosome conformation capture for genome scaffolding
- Short-read and/or long-read WGS for contig assembly
Data Analysis
- Hi-C-assisted chromosome-scale genome assembly scaffolding
- Transcriptomic marker profiling across forensically relevant developmental stages
- PMI estimation model building from molecular timeline data
Results
- Hi-C-Scaffolded Chromosome-Scale Genome Assembly
- Hi-C Library Construction and Sequencing from CD Genomics provided the chromatin contact data needed to scaffold the P. regina genome to chromosome scale, enabling reliable gene-level annotation essential for downstream transcriptomic analyses.
- The chromosome-scale assembly resolved repetitive regions characteristic of dipteran genomes, providing a high-quality reference for developmental marker identification.
- Molecular Marker Identification for Forensic PMI Estimation
- Using the scaffolded genome as reference, transcriptomic profiling identified stage-specific molecular markers across the blow fly developmental timeline — from egg through late larval and pupal stages.
- These markers enabled construction of a PMI estimation model based on gene expression patterns, providing forensic scientists with a molecular-level alternative to morphology-based developmental staging.
Hi-C-assisted chromosome-scale genome assembly and developmental molecular marker profiling in Phormia regina — enabling molecular PMI estimation for forensic applications. (PLOS Genetics, 2025, DOI: 10.1371/journal.pgen.1011948)
Conclusion
This study demonstrates the direct utility of Hi-C Library Construction and Sequencing for chromosome-scale genome assembly in forensically and ecologically important insects. The approach mirrors the core CiFi use case: a single insect species with complex repetitive genomic regions, limited tissue availability, and a need for high-quality chromosome-scale assembly to enable downstream molecular research. CiFi extends this capability further — enabling the same genome assembly workflow from input as low as a single individual, without pooling specimens.
Reference
- Unraveling forensic timelines using molecular markers in Phormia regina maggots. PLOS Genetics. 2025. https://doi.org/10.1371/journal.pgen.1011948
Related Publications
Here are some publications from our clients that used our Hi-C Library Construction and Sequencing service:
Unraveling forensic timelines using molecular markers in Phormia regina maggots
Journal: PLOS Genetics
Year: 2025
See more articles published by our clients.
