Fiber Array Sequencing Test Method

Fiber-seq is a single-molecule, long-read sequencing method that maps chromatin structure, DNA methylation, and protein occupancy on individual DNA fibers.Overview

Fiber-seq is a multiomic assay designed to capture both genomic and epigenomic information at single-molecule resolution. Unlike traditional short-read chromatin assays, Fiber-seq simultaneously provides data on DNA sequence, chromatin accessibility, nucleosome positioning, CpG methylation, and transcription factor binding on individual chromatin fibers, enabling haplotype-specific insights into gene regulation and chromatin dynamics .

Key Principles

• Enzymatic Labeling: The method uses the non-specific N6-adenine methyltransferase Hia5 to methylate adenines in accessible (unprotected) DNA regions. This 6mA mark is not naturally present in most eukaryotic genomes, allowing clear distinction from endogenous 5mC methylation .
• Single-Molecule Long-Read Sequencing: Labeled DNA is extracted and prepared using PacBio SMRTbell library protocols for HiFi sequencing. Each read represents a multi-kilobase chromatin fiber, capturing both the DNA sequence and epigenetic modifications .
• Protein Footprinting: Regions where adenines remain unmethylated indicate protein binding, and the size of these unmethylated footprints can infer the type of bound protein .

Workflow

1. Nuclei Isolation: Cells are harvested, and nuclei are extracted to preserve chromatin structure .
2. Hia5 Methylation Reaction: Nuclei are incubated with Hia5 and S-adenosylmethionine (SAM) to label accessible adenines with 6mA .
3. DNA Extraction and Library Preparation: High molecular weight DNA is purified and converted into SMRTbell libraries suitable for PacBio long-read sequencing .
4. Sequencing: PacBio HiFi sequencing detects both 6mA and endogenous 5mC modifications, generating highly accurate long reads representing individual chromatin fibers .
5. Data Analysis: Bioinformatic pipelines, such as Fibertools, are used to perform quality control, map methylation and accessibility, and infer regulatory elements and nucleosome positions .

Applications

• Chromatin Accessibility Mapping: Identifies open chromatin regions at near base-pair resolution.
• Epigenetic Profiling: Simultaneously captures DNA methylation patterns and protein occupancy.
• Haplotype-Resolved Analysis: Enables phasing of genetic variants and epigenetic features along individual DNA fibers.
• Regulatory Element Characterization: Provides insights into transcription factor binding, nucleosome positioning, and cis-regulatory architecture .

Advantages

• Single Assay Multiomic Data: Combines information that would otherwise require multiple separate assays (e.g., WGS, WGBS, ATAC-seq).
• High Resolution: Near single-nucleotide resolution on multi-kilobase DNA fibers.
• Reduced False Positives: Direct labeling of accessible DNA avoids enzymatic digestion artifacts common in other chromatin assays . Fiber-seq represents a cutting-edge approach in chromatin biology, offering a comprehensive view of genome regulation at the level of individual DNA molecules.