Fiber-seq and Hia5

EpiCypher Fiber-seq and Hia5 banner image with logos and product imagery on blue background

Fiber-seq – Multiple Assays in One

What is Fiber-seq?

EpiCypher’s CUTANA™ Fiber-seq kit is a breakthrough approach in multiomic long-read sequencing (LRS) that simultaneously maps genetic and epigenetic features from individual chromatin fibers. The Fiber-seq assay profiles chromatin accessibility, DNA methylation, and genetic variants. This single assay gives insights that would normally require integrating data from multiple methods such as ATAC-seq, ChIP-seq, and bisulfite sequencing.

The CUTANA™ Fiber-seq kit includes reagents to go from cells to purified genomic DNA that can be used directly in either Pacific Biosciences® (PacBio® HiFi Sequencing) or Oxford Nanopore Technologies® (ONT® Nanopore Sequencing) library preparation workflows. The recommended input for Fiber-seq is 1,000,000 native human nuclei per reaction.

 

Figure 1 image

Figure 1: Fiber-seq simultaneously detects open chromatin (marked by 6mA) and DNA methylation (5mC) in human leukemia (K562) cells. ATAC-seq and CTCF CUT&RUN data are shown for comparison. Each horizontal line in the Fiber-seq panels represents a single long read, providing single-molecule data.
Data from 60 individual DNA molecules are shown. 

Key Advantages

The CUTANA™ Fiber-seq Kit provides key reagents and a comprehensive manual to perform Fiber-seq.

  • Multiomic Insights in One Assay – Gain insights that would normally require integrating data from multiple methods such as ATAC-seq, ChIP-seq,
        and bisulfite sequencing.
  • Comprehensive Solution – This kit includes key reagents needed to go from cells to library prep-ready DNA.
  • User-friendly Resources – The User Manual is complete with a detailed protocol, QC checks, FAQs, and expert tips to get you started.
  • Easy Workflow Integration – Fiber-seq can be performed in <2 hours directly upstream of routine long-read sequencing library preparation and uses
        reagents vetted for compatibility with library prep kits from major LRS platforms.
  • Powered by Hia5 6mA methyltransferase – Hia5 is the highest activity 6mA-methyltransferase available and is exclusive to CUTANA™ Fiber-seq Kits. 
  • Stringent Quality Control – Each new lot undergoes rigorous quality control checks to ensure components yield consistent results in your research.

    FAQs

    How Does Fiber-seq Work?

    Fiber-seq involves three main steps:

    Step 1: Hia5 labeling

    • Isolated nuclei are incubated with the Hia5 enzyme for ~10 minutes.
    • Hia5 introduces 6mA marks at accessible DNA sites, enabling open chromatin mapping.

    Step 2: Long-read sequencing

    • Libraries are prepared for PacBio HiFi or ONT platforms (PCR-free workflows only).
    • Sequencing detects both Hia5-labeled 6mA and endogenous 5mC sites.

    Step 3: Data analysis

    • Data is analyzed using the fibertools pipeline to infer nucleosome positioning, transcription factor binding, and chromatin accessibility.

    Where Can I Find The EpiCypher Fiber-seq Protocol?

    The EpiCypher Fiber-seq protocol is available here. If you run into difficulties with your assay, please reach out to us at tech@chromspec.com! 

    What Samples and Cell Types Are Compatible with Fiber-seq?

    Fiber-seq is compatible with a wide range of inputs and has been successfully applied to human cell lines, primary cells, plants, yeast, and non-model organisms. The key requirement is that DNA be free of endogenous N6-methyladenosine as Fiber-seq relies on detecting 6mA, introduced during the assay by Hia5, to reveal open chromatin profiles. Thus, species that endogenously methylate their DNA (like most bacteria) are not compatible with Fiber-Seq.

    Fresh nuclei provide the best results, though flash-frozen and cross-linked samples are also compatible, with minor labeling differences. Sample quality and genome size are important factors that influence input requirements and overall data quality.

    Sample Input

    • Recommended: 1,000,000 fresh nuclei* per reaction. To account for sample loss, start with 2M cells with the goal of retaining 1M nuclei. If you are using non-human cells, the target number of nuclei may be different.
    • Flash-frozen and crosslinked nuclei are compatible (with slight labeling differences).
    • Harvest ~2M cells* per reaction with 10% extra if possible to account for sample loss.

    Currently, inputs <1M nuclei are not validated. Scaling (ie. decreasing Hia5 concentration for lower cell/nuclei inputs) is theoretically possible but not recommended at this time. 

    Does Hia5 Labeling Affect Sequencing Quality?

    No, Hia5 labeling does not affect sequencing quality. 6mA labeling is a non-destructive reaction and as such read lengths remain unchanged compared to untreated DNA. You may notice slight differences in read quality, but these are due to basecaller performance rather than any damage to the DNA.

    Can Fiber-seq Be Used to Study DNA-binding Proteins?

    Yes, Fiber-seq provides near-nucleotide resolution of chromatin accessibility, enabling detection of protein footprints.

    Important Notes:

    • Success depends on protein binding dynamics, occupancy, and footprint size.
    • Multiple labs have successfully published on TF and polymerase footprinting using Fiber-seq.
    • EpiCypher recommends using fibertools and fiberseq-qc for Fiber-seq data analysis.

    Check out these papers for real-world examples:

    What Library Prep Kits and Sequencing Machines Are Compatible with Fiber-seq?

    Fiber-seq is compatible with native DNA long-read sequencing workflows on both PacBio and Oxford Nanopore Technologies (ONT) platforms. For library prep, PacBio HiFi WGS Kits and ONT’s Ligation Sequencing Kit V14 are recommended. The method is not compatible with PCR-based workflows, but otherwise integrates seamlessly with either sequencing system.

    For users interested in performance comparisons between sequencing platforms, direct benchmarking data is available on GitHub.

    What If My Sample Is From an Organism Without a Reference Genome?

    Fiber-seq works with or without a high-quality reference genome for your organism. This is because long-read sequencing enables accurate, contiguous genome assemblies of novel model organisms and does not rely solely on alignment to a pre-existing genome to map reads. Fiber-seq allows you to capture chromatin features at the same time you’re constructing your genome assembly, enabling high-resolution, reference-free insights into gene regulation in emerging model systems.