Long-read Pore-C Sequencing Service

Comprehensive Workflow & QC Standards

We adhere to a rigorous, standardized workflow designed to maximize "valid multi-way contacts" while minimizing noise. Unlike "black box" service providers, we define clear QC checkpoints to ensure your data is interpretation-ready.

Step 1: In-Situ Chromatin Fixation & Permeabilization

The process begins with crosslinking cells using formaldehyde to "freeze" the 3D chromatin structure in place. We optimize lysis conditions to permeabilize the nuclei while keeping the nuclear envelope intact. This "in-situ" approach minimizes random ligation events (noise) that can occur in solution.

Step 2: Restriction Digestion

We utilize optimized restriction enzyme cocktails (e.g., frequent cutters like NlaIII or DpnII, or 4-cutter/6-cutter combinations depending on the genome density required).

• Why it matters: The choice of enzyme determines the theoretical resolution. For genome assembly, a specific enzyme strategy helps generate uniform distribution of cut sites across the genome, ensuring no regions are left unscaffolded.

Step 3: Proximity Ligation (The Critical Difference)

In standard Hi-C, ligation is performed to join two ends. In Pore-C, ligation conditions are optimized to encourage multiple restriction fragments to ligate together into long chains (concatemers).

• Technical Note: We do not perform biotin enrichment or sonication at this stage. This preserves the full length of the ligated concatemer.

Step 4: Protein Degradation & DNA Purification

Crosslinks are reversed, and proteins are degraded using Proteinase K. The resulting high-molecular-weight (HMW) DNA consists of chimeric sequences—mosaics of genomic regions that were spatially proximal. Gentle handling is crucial here to prevent shearing the long DNA molecules.

Step 5: Nanopore Library Preparation & Sequencing

The HMW DNA is prepared for ONT sequencing using ligation sequencing kits (e.g., LSK series). We load the library onto PromethION or GridION flow cells to ensure sufficient depth.

• Target Depth: While depth varies by genome size, we aim for sufficient coverage to call meaningful multi-way contacts. (Note: Specific Gb output is project-dependent; please Request a Quote for estimates).

Step 6: Rigorous Quality Control (QC)

We report transparent QC metrics mandated by our strict internal standards:

• N50 Read Length: Confirms preservation of long molecules.
• Concatemericity: The average number of restriction fragments per read. A high "fragments-per-read" count indicates successful multi-way capture.
• Cis/Trans Ratio: Monitoring the ratio of intra-chromosomal vs. inter-chromosomal contacts to assess library signal-to-noise.
• Methylation Call Rates: Verifying the detection of 5mC modifications against expected baselines.

Applications: Unlocking New Dimensions of the Genome

Long-read Pore-C Sequencing Service is not just an alternative to Hi-C; it is an enabler for specific high-difficulty research questions.

1. Decoding Enhancer-Promoter Hubs (V2G)

Genome-Wide Association Studies (GWAS) often identify risk variants in non-coding regions. The challenge is linking these variants to the genes they regulate, which may be megabases away.

• The Hub Hypothesis: Enhancers often function in clusters. Pore-C allows you to see if a risk variant, an enhancer, and a gene promoter are all part of the same physical complex.
• Validation: This provides stronger evidence for Variant-to-Gene (V2G) assignment than simple correlation, prioritizing targets for drug discovery.

2. Plant & Animal Genome Assembly

Many plant genomes are polyploid and highly repetitive. Short reads cannot distinguish between identical repeat copies on different chromosomes.

• Scaffolding: Pore-C reads, being long, can anchor unique sequences on either side of a repeat tract, effectively "bridging" the gap.
• Phasing: Because the reads are long enough to contain multiple heterozygous SNPs, Pore-C can phase entire chromosomes (separating maternal and paternal haplotypes) with high accuracy.

3. Structural Variation (SV) Detection

In cancer genomics, chromosomes often undergo massive rearrangements (translocations, inversions).

• The Problem: Short-read Hi-C often yields "noisy" signals at breakpoints.
• The Pore-C Solution: A Pore-C read can physically span the breakpoint junction. Sequencing the junction directly confirms the exact fusion point, distinguishing true SVs from ligation artifacts.

Frequently Asked Questions (FAQ)