PacBio Webinar: Small Samples, Big Insights - Capturing Chromatin Architecture with CiFi
One fly, three library types: chromosome-scale assembly with Fiber-seq and CiFi
Long-read low-pass for polyploid crops: 32% more variants than short-reads
Hi-C characterizes three-dimensional chromatin organization, facilitates haplotype phasing, and enables genome-assembly scaffolding, but encounters difficulties across complex regions. By coupling chromosome conformation capture (3C) with PacBio HiFi long-read sequencing, here we develop a method (CiFi) that enables analysis of genomic interactions across repetitive regions. Starting with as little as 60,000 cells (sub-microgram DNA), the method produces multi-kilobasepair HiFi reads that contain multiple interacting, concatenated segments (~350 bp to 2 kbp). This multiplicity and increase in segment length versus standard short-read-based Hi-C improves read-mapping efficiency and coverage in repetitive regions and enhances haplotype phasing. CiFi pairwise interactions are largely concordant with Hi-C from a human lymphoblastoid cell line, with gains in assigning topologically associating domains across centromeres, segmental duplications, and human disease-associated genomic hotspots. As CiFi requires less input versus established methods, we apply the approach to characterize single small insects: assaying chromatin interactions across the genome from an *Anopheles coluzzii* mosquito and producing a chromosome-scale scaffolded assembly from a *Ceratitis capitata* Mediterranean fruit fly. Together, CiFi enables assessment of chromosome-scale interactions of previously recalcitrant low-complexity loci, low-input samples and small organisms.
Step 1: CiFi starts with an in situ 3C/Hi-C-style proximity ligation: chromatin is cross-linked, cut with a restriction enzyme, and nearby DNA fragments are ligated together, then crosslinks are reversed and DNA is purified.
Step 2: CiFi boosts yield from low input by adding a high-fidelity, genome-wide amplification step and preparing a PacBio SMRTbell library for HiFi sequencing (with size selection), enabling long multi-contact reads from as little as ~60,000 cells.
Step 3: The long HiFi concatemers are split in silico at restriction sites into multiple interacting segments, which are converted into chromatin contacts for downstream 3D genome analyses.
What makes CiFi different
CiFi couples 3C with PacBio HiFi sequencing plus unbiased whole-genome amplification, generating multi-contact reads from as little as ~60,000 cells (sub-µg DNA).
By producing longer multi-kilobase segments and many interactions per read, CiFi improves mapping and coverage in repetitive regions (e.g., centromeres and segmental duplications) and substantially boosts haplotype phasing compared with short-read Hi-C, enabling stronger domain/TAD analysis across complex hotspots.
CiFi demonstrates practical end-to-end utility at small scales, profiling genome-wide chromatin interactions from a single Anopheles coluzzii mosquito and producing chromosome-scale scaffolding (phased diploid assembly) for a single Ceratitis capitata fruit fly—at competitive or lower contact requirements than traditional Hi-C.
The team behind CiFi
USDA Agricultural Research Service
Two CiFi library preparation protocols are now publicly available on protocols.io. The Arima 3C with PacBio HiFi Sequencing (CiFi) Library Preparation Protocol and the standard CiFi: 3C Library Preparation for PacBio HiFi Sequencing protocol provide step-by-step guidance for generating long-read chromatin conformation capture libraries compatible with PacBio HiFi sequencing.
Read moreA new strategy combines PacBio HiFi and CiFi sequencing in a single library to produce haplotype-resolved, chromosome-scale diploid assemblies — applied here to prairie and meadow vole genomes. The approach simplifies and reduces the cost of genome assembly, producing near-complete genomes using one sequencing platform.
Read morePacBio announces CiFi, a new long-read 3C method developed with UC Davis researchers that enables chromosome-scale assemblies from a single SMRT Cell. The method combines chromosome conformation capture with HiFi sequencing for low-input chromatin analysis.
Read moreScott Geib (USDA-ARS) presented CiFi at the Plant and Animal Genome Conference 2025 as part of the PacBio Workshop series. The talk demonstrated CiFi's capabilities for low-input, long-read chromosome conformation capture and its applications in genomics research.
Read moreHave questions about CiFi or need help with implementation? We'd love to hear from you.
