---
title: "How does Fiber-seq work?"
slug: "how-does-fiber-seq-work"
updated: 2025-11-07T15:37:00Z
published: 2025-11-11T13:00:01Z
canonical: "support.epicypher.com/how-does-fiber-seq-work"
---

> ## Documentation Index
> Fetch the complete documentation index at: https://support.epicypher.com/llms.txt
> Use this file to discover all available pages before exploring further.

# 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. For more on data analysis, see [this article](/v1/docs/how-do-i-analyze-fiber-seq-data).

![Steps of Fiber-seq.](https://cdn.document360.io/1fc69f91-cf1e-45ab-bc51-4063eb80e37c/Images/Documentation/fiberseqsteps.png)

Figure 1: Schematic of the CUTANA™ Fiber-seq workflow. **Step 1:** Hia5 methyltransferase labels accessible chromatin regions by methylating adenines (6mA) during a 10-minute enzyme incubation. **Step 2:** DNA is prepared for long-read sequencing using either PacBio or Nanopore (ONT) platforms. **Step 3:** Sequencing and analysis provide single-molecule resolution maps of chromatin accessibility, endogenous DNA methylation, and genetic variants in a single assay.