---
title: "How can I figure out the ideal Hia5 ratio for my project?"
slug: "how-can-i-figure-out-the-ideal-hia5-ratio-for-my-project"
updated: 2026-05-12T12:00:01Z
published: 2026-05-12T12:00:01Z
canonical: "support.epicypher.com/how-can-i-figure-out-the-ideal-hia5-ratio-for-my-project"
---

> ## 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 can I figure out the ideal Hia5 ratio for my project?

In human cells, we have found that the Hia5:nuclei ratio detailed in the Fiber-seq [manual](https://www.epicypher.com/wp-content/uploads/fiber-seq-kit-manual_v1.pdf) is ideal for obtaining optimal 6mA labeling (~6% labeling across the entire human genome). However, when working with a new species or unique conditions, it may be worthwhile to run an optimization experiment to experimentally determine this ratio in your sample type, as outlined below:

### Recommended Workflow

#### 1. Perform a 3-Point Cell Titration

To determine the optimal conditions for 6mA labeling, set up three parallel Fiber-seq reactions using varying nuclei inputs:

- **500,000 nuclei** (or 0.5× the standard input based on your cell’s genome—see more on standard inputs [here](/v1/docs/what-samples-and-cell-types-are-compatible-with-fiber-seq))
- **1,000,000 nuclei** (or the standard input based on your cell’s genome)
- **2,000,000 nuclei** (or 2× the standard input based on your cell’s genome)

This range allows you to assess how input quantity impacts labeling efficiency and overall data quality.

#### 2. Run Fiber-seq Reactions

Process each condition independently through the full Fiber-seq workflow, including library prep. Be sure to keep all other variables consistent across reactions to ensure that differences in output are attributable to input amount.

#### 3. Perform Shallow Sequencing

Sequence each sample at approximately **~1× coverage**. This level of sequencing is sufficient to evaluate 6mA labeling efficiency without committing to a full-depth run. For more on QC sequencing, see our section on it [here](http://support.epicypher.com/fiber-seq/shallow-qc-sequencing-for-fiber-seq).

#### 4. Evaluate % 6mA Labeling

Analyze the sequencing data to determine the **percentage of 6mA incorporation** for each condition. This metric serves as a key indicator of labeling efficiency.

- Identify the input condition that provides **optimal labeling** (e.g., strong signal without over-labeling or signal saturation—in human cells, EpiCypher finds that 6% labeling achieves these goals).
- Consider overall data quality in addition to % 6mA when selecting conditions.

#### 5. Proceed with Final Experiment

Once the optimal input amount has been determined, use those conditions for your full-scale Fiber-seq experiment.

### Additional Considerations

- **Limited or precious samples:** If your sample availability is constrained, consider performing the titration using a **closely related cell type**. This can provide a useful approximation for optimizing conditions while preserving your primary material.
- **Enzyme titration option:** In addition to varying cell input, you may also perform a titration of the **Hia5 methyltransferase concentration**:

This can help fine-tune labeling efficiency, particularly if cell input is fixed or limited.
  - 0.5× standard concentration
  - 1× standard concentration
  - 2× standard concentration

By incorporating a brief titration step into your workflow, you can significantly improve the reliability and interpretability of your Fiber-seq results.