Q1 How does ACE-seq compare to traditional bisulfite sequencing (BS-seq) or oxidative BS-seq (oxBS-seq)?

BS-seq cannot distinguish 5mC from 5hmC. OxBS-seq can, but it requires two separate libraries, doubles cost/input and causes extreme DNA fragmentation. ACE-seq uses a gentle enzyme to specifically detect 5hmC in one library, offering superior accuracy with far less DNA.

Q2 What is the minimum recommended sequencing depth for ACE-seq?

For mammalian genomes, we typically recommend 20-30x coverage for robust 5hmC detection. Depth may be adjusted based on genome size and specific project aims (e.g., focused on promoters vs. whole genome).

Q3 Can ACE-seq be applied to non-mammalian species?

Yes, ACE-seq is applicable to any organism with a cytosine-based epigenome. Protocol optimization may be required, and we welcome collaborative inquiries.

Q4 Do you provide differential analysis between multiple sample groups?

Absolutely. Our standard bioinformatics package includes DhMR identification and associated functional enrichment analysis for comparative studies.

Q5 How can ACE-seq help reduce false positives in differential 5hmC analysis?

We reduce false positives by combining upfront study-design guidance (replicates, covariates, batch control) with transparent, multi-stage QC and standardized filtering/statistical thresholds. Deliverables include QC metrics plus evidence plots (tracks/heatmaps/volcano) so differential 5hmC calls are easier to validate and interpret (RUO).

ACE Sequencing: Unlock the Epigenome with Single-Base Resolution 5hmC Profiling

Single-base 5hmC profiling for researchers who need confident, reproducible epigenomic signals—without guesswork.

From sample QC and optimized library construction to bioinformatics and interpretation, we help you pinpoint differential 5hmC regions and link them to genes and pathways (RUO).

Get a clear plan for inputs, depth, replicates, and expected outputs—so your project is designed right from day one.

True single-base 5hmC mapping with transparent QC metrics and conversion reporting
Study-design support (replicates, depth, confounders) to reduce rework and failed comparisons
Interpretation-ready deliverables: tracks, tables, and publication-style plots you can use immediately

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illustrates of ACE-seq is a method for detecting 5hmC methylation.

Introduction

Service Overview

ACE Sequencing (APOBEC-Coupled Epigenetic Sequencing, ACE-seq) is an enzymatic, genome-wide approach for mapping 5-hydroxymethylcytosine (5hmC) at single-base resolution. It supports differential 5hmC analysis across conditions or cohorts and helps interpret hydroxymethylation changes in regulatory elements and gene bodies.

5hmC is an epigenomic mark widely studied in developmental regulation, cellular differentiation, aging-related epigenomic remodeling, and disease-mechanism research. With CD Genomics, you receive an end-to-end workflow—from sample QC to bioinformatics and reporting—designed to deliver reproducible, interpretation-ready outputs.

Technologies

Cost-Effective Sequencing for Single-Base Resolution 5hmC Profiling

ACE Sequencing is a revolutionary NGS-based method that specifically detects 5-hydroxymethylcytosine. It utilizes the engineered enzyme APOBEC3A (A3A) to deaminate unmodified C and 5mC to U, while 5hmC—protected by glucosylation—remains as C. This clever biochemical trick allows precise discrimination and quantification of 5hmC without the harsh chemicals used in bisulfite sequencing.

The schematic diagram of the technical principle is as follows:

1）β-Glucosyltransferase (βGT) selectively glucosylates 5hmC to form 5ghmC, which is protected from A3A deamination.

2）The A3A enzyme specifically deaminates unmodified C and 5mC to U, while 5hmC remains as C, enabling its specific detection.

illustrates of core workflow of the ACE-seq technique for detecting 5hmC (5-hydroxymethylcytosine)

Comparison of several genome-wide methylation methods：

Method	Principle	Target Modification(s)	Single-Base Resolution	Key Advantages
ACE-Seq	Enzymatic deamination (APOBEC3A selectively deaminates C/5mC, but not 5hmC)	5hmC	Yes	Minimal DNA damage; low input requirement; bisulfite-free
TAB-Seq	βGT glucosylation of 5hmC → TET oxidation of 5mC → bisulfite conversion	5hmC	Yes	High specificity; considered a gold standard for 5hmC mapping
oxBS-Seq	KRuO₄ oxidation of 5hmC to 5fC → bisulfite conversion	5mC (5hmC inferred by subtraction from BS-Seq)	Yes	Enables quantitative separation of 5mC and 5hmC
EM-Seq	TET2 oxidation of 5mC → 5caC; APOBEC deamination of unmodified C (± βGT for 5hmC)	5mC (standard); 5mC + 5hmC (with βGT pre-treatment)	Yes	low DNA damage; ultra-low input compatible; compatible with long-read sequencing

Why Choose ACE Sequencing?

While traditional 5hmC profiling methods like oxBS-Seq are hampered by high cost, substantial DNA input requirements, and damaging chemical treatment, ACE Sequencing provides an elegant solution. Our technology delivers:

Single-Base Resolution: Map 5hmC across the entire genome with base-by-base precision, uncovering regulatory details missed by other methods.
Ultra-Low Input: Requires less DNA than oxidative bisulfite methods, enabling analysis of precious, limited, or degraded samples.
Superior Data Fidelity: Our gentle enzymatic conversion preserves DNA integrity, yielding higher-quality libraries and more reliable, accurate data.
Ideal for Challenging Samples: Unlock insights from rare specimens, FFPE tissues, and cell-free DNA where material is scarce.
Future-Proof Your Research: Gain the definitive, most precise view of the dynamic hydroxymethylome to build a robust foundation for discovery.

Applications

Applications of ACE Sequencing

ACE Sequencing empowers discovery across diverse biological fields:

Discovering Disease Biomarkers

ACE Sequencing detects disease-specific 5hmC "signatures" that appear early in tissues and liquid biopsies (e.g., cfDNA). These highly sensitive epigenetic biomarkers support biomarker discovery, cohort stratification, and mechanism studies using tissue or cfDNA in research settings.

Decoding Development & Cell Fate

ACE Sequencing maps 5hmC dynamics at single-base resolution during critical processes like embryogenesis and stem cell differentiation. By pinpointing 5hmC at gene regulators, it reveals how this modification acts as a precise epigenetic switch controlling cell-specific gene expression, providing foundational insights for developmental biology and regenerative medicine.

Revealing Environmental Adaptation

This application studies how organisms rapidly reprogram their 5hmC landscape in response to stresses like drought or disease. ACE Sequencing identifies the specific genes and pathways regulated by these epigenetic changes, offering direct targets for breeding more resilient crops and improving animal health management.

Enhancing Key Economic Traits

ACE Sequencing discovers stable 5hmC patterns linked to superior agricultural traits (e.g., yield, growth rate). These epigenetic markers enable novel, non-genetic breeding strategies, accelerating the development of improved plant varieties and animal breeds through precise trait optimization.

Service workflow

ACE Sequencing Service: Step-by-Step Process

Bioinformatics

Bioinformatics Analysis

CD Genomics offers comprehensive and flexible bioinformatics analysis services, ranging from basic data processing to advanced customized analyses. Our solutions facilitate in-depth exploration of genomic variations and functionalities.

Standard Analysis

Raw Data QC and Alignment to Reference Genome
Genome-Wide 5hmC Site Detection and Quantification
Visualization of 5hmC Distribution (e.g., across genes, promoters, enhancers)
Differential Hydroxymethylated Region (DhMR) Analysis
Gene Ontology (GO) and Pathway (KEGG) Enrichment Analysis of Target Genes
Sample Clustering and PCA Analysis

Advanced & Custom Analysis:

Multi-omics Integration (e.g., with RNA-seq or ChIP-seq data)
Correlation with Clinical Phenotypes
Customized Analysis per Your Specific Research Goals

Bioinformatics analysis workflow for ACE-seq sequencing data

Demo

The following figures display a portion of the data.

Genome-wideHydroxymethylation Profile

Landscape of genome-wide hydroxymethylation

hydromethylation level across genomic features/functional genomic elements

Giros plot of sample-specific hydroxymethylaition

correlation analysis of genome-wide hydeoxymethylation profiles

base-resolution density of hydeoxymethylated cytosines per chromosome

Sample Requirements

Sample Requirements and Preparation

Sample Type	Minimum Amount	Quality Notes
Genomic DNA	≥ 100 ng	High purity (A260/280 ≈ 1.8), in TE or water.
Fresh/Frozen Tissue	≥ 10 mg	Snap-frozen, avoid RNAlater.
Cultured Cells	≥ 2 x 10⁶ cells	Pelleted, washed with PBS.
Whole Blood	0.5–2 mL	EDTA or citrate tubes preferred.
FFPE Sections	≥ 15 slides (4-10 µm)	Tumor cell purity >30% recommended.
Plasma/Serum cfDNA	≥ 15 ng cfDNA	Isolated using recommended kits.
Other Types	Please Inquire	Saliva, urine, microbiome DNA, etc.

Shipment: Ship samples on dry ice. Contact us for detailed preparation guidelines.

Advantage

Why Choose CD Genomics for ACE Sequencing?

Choosing a service provider is about partnership, expertise, and guaranteed quality. At CD Genomics, we combine cutting-edge technology with unwavering scientific rigor to ensure your project's success.

1. Specialized Epigenetics Expertise:

Our scientists are not just sequencing technicians; they are experts in epigenetic assay development and optimization. We understand the nuances of 5hmC biology and data interpretation, providing you with insightful consultation from project design to discussion.

2. End-to-End Project Ownership with Dedicated Support:

You will be assigned a dedicated project manager and a team of expert analysts. This single point of contact ensures clear communication, timely updates, and seamless coordination across our wet-lab and bioinformatics teams, giving you peace of mind.

3. Rigorous, Multi-Stage Quality Control:

Quality is embedded at every step. We perform stringent QC on incoming samples, monitor conversion efficiency post-enzymatic treatment, assess library quality, and validate data output. Our transparent QC reports give you confidence in the foundational data driving your discoveries.

4. State-of-the-Art Infrastructure & Proven Protocols:

We run projects on calibrated Illumina NovaSeq & NextSeq systems. Our ACE-seq workflow is optimized and routinely monitored with spike-in controls and reference samples for consistent performance across projects.

5. Actionable Data Delivery, Not Just Raw Output:

We deliver more than just FASTQ files. Our analysis pipeline is curated to yield biologically meaningful results, presented in clear, publication-ready figures and comprehensive reports. We focus on turning data into your next discovery.

6. Collaborative & Flexible Approach:

We view each project as a collaboration. Whether you need a standard whole-genome profile or a custom-targeted panel, our team works flexibly to tailor the service to your specific hypotheses, budget, and sample types.

FAQ

Frequently Asked Questions (FAQ)

Q6 I have limited samples or budget—how should I design a "pilot-to-scale" 5hmC study with ACE-seq?

References

Schutsky, E.K., DeNizio,j.E. et al. Nondestructive, base-resolution sequencing of 5-hydroxymethylcytosine using a DNA deaminase. Nat Biotechnol 36, 1083–1090 (2018).
Wu, X., Zhang, Y. et al. TET-mediated active DNA demethylation: mechanism, function and beyond. Nat Rev Genet 18, 517–534 (2017).
Yan,R.,Cheng,X. et al. Dynamics of DNA hydroxymethylation and methylation during mouse embryonic and germline development. Nature Genetics 55,130-143 (2023).
Chen X.F.,Xu H.Q., et al. Chun-Xiao Song Mapping epigenetic modifications by sequencing technologies. Cell Death Differ 32(1), 56-65 (2025).