oxBS-seq Services - Single-Base 5hmC Mapping

Overview

Overview of Our oxBS-seq Services

5-hydroxymethylcytosine (5hmC) is the oxidized derivative of 5-methylcytosine (5mC), generated by TET family dioxygenases during active DNA demethylation. Unlike 5mC — typically associated with transcriptional repression — 5hmC is enriched at active enhancers, gene bodies, and tissue-specific regulatory regions, where it marks dynamic epigenetic regulation. Standard bisulfite sequencing (BS-seq) cannot distinguish 5hmC from 5mC: both modifications resist bisulfite conversion and are read as cytosine, conflating two functionally distinct marks.

Oxidative bisulfite sequencing (oxBS-seq) solves this problem. The method, first described by Booth et al. (Science, 2012), adds a selective chemical oxidation step before bisulfite conversion. Potassium perruthenate (KRuO₄) specifically oxidizes 5hmC to 5-formylcytosine (5fC), which bisulfite then converts to uracil — read as thymine during sequencing. 5mC remains unchanged throughout. By sequencing both a standard BS library (5mC + 5hmC) and an oxBS library (5mC only) from the same DNA sample, true 5hmC levels are derived by subtraction at single-base resolution.

At CD Genomics, we offer an integrated oxBS-seq platform covering three service tiers — oxWGBS, oxRRBS, and Targeted oxBS — each with paired BS and oxBS library construction, sequencing, and full bioinformatics support for 5hmC quantification.

Technologies

Core Technologies: oxWGBS, oxRRBS, and Targeted oxBS

oxWGBS — Whole-Genome Oxidative Bisulfite Sequencing

oxWGBS applies the oxidative bisulfite chemistry to whole-genome bisulfite sequencing, producing the most comprehensive 5hmC map available. Every CpG site covered by sufficient sequencing depth yields a quantitative 5hmC estimate. This is the method of choice for studies requiring an unbiased, genome-wide view of 5hmC distribution — including discovery of novel differentially hydroxymethylated regions (DhMRs), characterization of tissue-specific 5hmC landscapes, and multi-omics integration with matched transcriptomic or histone modification data.

Because oxWGBS covers the entire genome, it requires the highest sequencing depth per sample. Paired BS and oxBS libraries mean each sample is sequenced twice — a consideration for large cohort studies.

oxRRBS — Reduced Representation Oxidative Bisulfite Sequencing

oxRRBS combines MspI restriction enzyme digestion (which enriches for CpG-rich regions including CpG islands, shores, and promoters) with oxidative bisulfite conversion. By focusing sequencing on the most functionally relevant fraction of the genome, oxRRBS provides single-base 5hmC quantification at a fraction of the sequencing cost of oxWGBS.

oxRRBS is well-suited for:

Large cohort studies where oxWGBS would be cost-prohibitive
Focused analysis of promoter and CpG island 5hmC dynamics
Initial screening to identify candidate DhMRs for follow-up validation with targeted oxBS
Non-model organisms where reduced representation simplifies alignment and analysis

Targeted oxBS — Region-Specific 5hmC Validation

Targeted oxBS uses capture probes or amplicon-based enrichment to focus sequencing on predetermined genomic regions. This is the highest-resolution, most cost-efficient oxBS-seq option, ideal for:

Validating DhMRs identified by oxWGBS or oxRRBS screening
Profiling 5hmC at known regulatory regions across large sample cohorts
Clinical or translational studies where only specific loci are of interest

For broader methylation profiling needs, our Whole Genome Bisulfite Sequencing (WGBS) and RRBS services are also available.

Selection Guide

Method Selection Guide: Choosing the Right oxBS-seq Approach

The table below compares the three oxBS-seq service tiers across key dimensions to help you match the method to your research goals.

Criterion	oxWGBS	oxRRBS	Targeted oxBS
Genomic coverage	Whole genome, unbiased	CpG-rich regions (CpG islands, promoters, shores)	User-defined loci only
Resolution	Single-base	Single-base	Single-base
5mC quantification	Genome-wide, per CpG	Focused on CpG-rich regions	Targeted regions only
5hmC quantification	By subtraction (BS − oxBS), per CpG	By subtraction, per covered CpG in enriched regions	By subtraction, per targeted CpG
Sequencing depth required	Highest (×2 for paired libraries)	Moderate	Lowest (focused coverage)
Cost per sample	Highest	Moderate	Most cost-efficient
Non-model organism support	Requires reference genome	Reference genome recommended; reduced representation simplifies analysis	Requires known target sequences
Best for	Comprehensive 5hmC discovery; tissue-specific landscape mapping; multi-omics integration	Large cohort studies; promoter and CpG island 5hmC profiling; cost-effective screening	Validation of candidate regions; large-sample targeted studies; clinical/translational research

Selection Strategy:

Define your discovery scope. If you need an unbiased genome-wide 5hmC map and have a small number of samples, oxWGBS provides the most complete data. For large sample cohorts or studies focused on gene regulatory regions, oxRRBS offers a cost-effective alternative.
Consider the paired-library cost. Each sample in any oxBS-seq method requires two libraries (BS + oxBS) and twice the sequencing of a standard methylation study. oxRRBS and Targeted oxBS mitigate this by reducing the genomic fraction sequenced.
Match resolution to your question. All three methods deliver single-base resolution — the difference is in genomic breadth, not data quality at covered sites.
For non-model organisms. oxRRBS is often the most practical starting point, as the reduced representation simplifies alignment and the MspI enrichment is independent of prior genome annotation.

Speak with an oxBS-seq Specialist

Workflow

End-to-End Workflow and Quality Control

Our oxBS-seq service follows a standardized workflow with QC checkpoints at each stage. The defining feature is the paired BS and oxBS library preparation — two parallel paths that diverge at the oxidation step before converging on shared sequencing and analysis.

Sample Receipt and Quality Assessment — QC Checkpoint: DNA quantity (Qubit) and purity (OD260/280); genomic integrity by gel electrophoresis or Bioanalyzer/TapeStation; concentration verification.
Library Preparation — BS and oxBS in Parallel — QC Checkpoint (BS library): DNA fragmentation, end-repair, adapter ligation, bisulfite conversion. QC Checkpoint (oxBS library): DNA fragmentation, end-repair, adapter ligation, KRuO₄ oxidation of 5hmC to 5fC, bisulfite conversion. Oxidation efficiency monitored by spike-in controls at this step.
Sequencing — QC Checkpoint: Sequencing yield and Q30 scores; duplication rate; bisulfite conversion efficiency from read-level C→T rates at non-CpG cytosines; coverage assessment.
Data QC — QC Checkpoint: Alignment rates for both BS and oxBS libraries; coverage correlation between paired libraries; per-sample methylation summary statistics; replicate correlation.
Bioinformatics and Report Delivery — QC Checkpoint: 5hmC call reproducibility; differential analysis statistical metrics; deliverable completeness verification; final QC summary.

Sample

Sample Requirements and Preparation Guidelines

DNA quality is the single most important factor for successful oxBS-seq. Both bisulfite conversion and KRuO₄ oxidation contribute to DNA loss, so starting with intact, high-purity DNA is essential.

Service Tier	Recommended DNA Type	Input Guideline	Key QC Metrics	Notes
oxWGBS	High-quality gDNA	≥ 3 μg gDNA recommended; ≥ 1 μg minimum; ≥ 30 ng/μL	OD260/280: 1.8–2.0; concentration ≥ 50 ng/μL; no visible degradation	Paired BS + oxBS libraries require higher total input than standard WGBS; DNA loss occurs at both oxidation and bisulfite steps
oxRRBS	High-quality gDNA	≥ 2 μg gDNA recommended; ≥ 50 ng/μL	OD260/280: 1.8–2.0; concentration ≥ 50 ng/μL	MspI digestion enriches for CpG-rich regions independent of prior genome annotation; lower total input required than oxWGBS
Targeted oxBS	High-quality gDNA	≥ 1 μg gDNA recommended; ≥ 20 ng/μL	OD260/280: 1.8–2.0; concentration ≥ 50 ng/μL	Capture probe or amplicon-based enrichment; the most input-efficient option; suitable for large sample cohorts
oxBS-seq (FFPE)	FFPE-derived DNA	Project-specific — contact us	DV200 assessment recommended	Higher input needed to compensate for formalin-induced degradation; oxidative chemistry tolerates crosslink damage better than standard bisulfite — but DNA integrity still affects library complexity

All samples should be shipped on dry ice and stored at -80°C. Avoid repeated freeze-thaw cycles.

Bioinformatics

Bioinformatics Analysis and Deliverables

oxBS-seq generates paired datasets per sample, requiring specialized bioinformatics to process BS and oxBS libraries in parallel and derive 5hmC estimates. Our pipeline integrates established tools for bisulfite-aware alignment, methylation calling, and differential analysis.

Standard Deliverables:

Deliverable	Description
Raw sequencing data	Demultiplexed read files for BS and oxBS libraries with quality scores
Aligned reads	Bisulfite-aware genome alignment for each library
Methylation calls — BS library	Per-CpG methylation ratio (5mC + 5hmC) from the BS library
Methylation calls — oxBS library	Per-CpG true 5mC ratio from the oxBS library (5hmC oxidized and converted)
5hmC quantification	Per-CpG 5hmC level derived by BS − oxBS subtraction at each covered site
QC report	BS and oxBS conversion efficiency, alignment rates, coverage metrics, library correlation, replicate assessment
Differential 5hmC analysis	Statistically significant DhMRs and DhMSs between user-defined condition groups
Genomic annotation	DhMRs/DhMSs annotated to genes, promoters, CpG islands, enhancers, and other genomic features
GO/KEGG enrichment	Functional enrichment of genes associated with differential 5hmC

Optional Advanced Analysis:

Oxi-mC modeling — probabilistic deconvolution of 5mC, 5hmC, and unmethylated cytosine proportions at each CpG site, accounting for oxidation and conversion efficiencies
Multi-omics integration — combined 5hmC data with matched RNA-seq, ChIP-seq, or ATAC-seq to link hydroxymethylation changes to transcriptional and chromatin state alterations
cfDNA-specific 5hmC analysis — specialized processing for cell-free DNA samples, including fragment size-aware analysis and tissue-of-origin deconvolution
Custom visualization — publication-ready figures including genome browser tracks, heatmaps, volcano plots, and circos plots
Epigenomic data analysis — see our dedicated Epigenomic Data Analysis service

Demo

Representative Data and Demo Results

The composite image below illustrates the data types delivered with each oxBS-seq project. All panels represent standard bioinformatics output formats generated by our pipeline.

Single-Base 5hmC Resolution and Genomic Distribution:

BS/oxBS/5hmC methylation track (Panel A) — IGV-style genome browser view showing three parallel tracks: BS-seq (total 5mC + 5hmC signal), oxBS-seq (true 5mC only), and the subtraction track (5hmC) at single-base resolution. Each dot represents a CpG position; the subtraction at each position directly quantifies 5hmC stoichiometry.
5hmC enrichment heatmap (Panel B) — metaplot and heatmap showing average 5hmC signal distribution across gene bodies, CpG islands, and enhancer regions. Reveals the characteristic enrichment of 5hmC at active regulatory elements versus its depletion at silenced regions.
5mC vs 5hmC density scatter (Panel C) — per-CpG comparison of 5mC and 5hmC levels, showing the relationship between the two modifications genome-wide. The majority of CpGs carry 5mC only; sites with appreciable 5hmC are a distinct subpopulation concentrated at specific genomic compartments.

Differential Analysis and Functional Interpretation:

Differential 5hmC volcano plot (Panel D) — each point represents a CpG site; x-axis shows log2 5hmC fold change between conditions; y-axis shows -log10 adjusted p-value. Sites with significantly increased (hyper-hydroxymethylated, red) or decreased (hypo-hydroxymethylated, blue) 5hmC are highlighted.
Genomic feature distribution (Panel E) — breakdown of 5hmC sites across genomic compartments (promoters, enhancers, gene bodies, CpG islands, intergenic regions), providing a global view of where 5hmC is deposited in the genome.
GO/KEGG enrichment (Panel F) — top enriched biological process terms among genes associated with differentially hydroxymethylated CpGs, linking 5hmC changes to specific pathways and functional categories.

All demo results are generated from representative datasets and reflect the standard analysis depth delivered with each project. Actual figures are customized to your experimental design and research question.

Applications

Applications Across Research Areas

Cancer Epigenomics

5hmC is globally depleted across multiple cancer types, with redistribution at enhancers and tissue-specific regulatory regions. Profiling 5hmC in tumor versus matched normal tissue can identify cancer-specific DhMRs that serve as diagnostic or prognostic biomarkers. In cfDNA, tumor-derived 5hmC signatures are being explored for non-invasive early detection and disease monitoring (Rech et al., medRxiv, 2025).

Neuroscience and Neurodegeneration

The brain exhibits the highest 5hmC levels of any tissue, reflecting the elevated expression of TET enzymes in neurons. 5hmC dynamics are implicated in learning, memory, aging, and neurodegenerative diseases including Alzheimer's and Huntington's. oxBS-seq provides the single-base resolution needed to map 5hmC at individual regulatory elements in specific brain regions and cell types.

Developmental and Stem Cell Biology

Active DNA demethylation via TET-mediated 5mC oxidation to 5hmC is essential for epigenetic reprogramming during early embryogenesis and germ cell development. oxBS-seq can track 5hmC dynamics across developmental time courses, revealing the kinetics of demethylation at specific regulatory elements during lineage commitment and cell fate transitions.

Liquid Biopsy and cfDNA Biomarker Discovery

cfDNA 5hmC is emerging as a promising biomarker class for non-invasive cancer detection. Unlike mutations, 5hmC patterns carry tissue-of-origin information, and tumor-specific 5hmC signatures can be detected in plasma. A 2024 review in Genes comprehensively surveyed oxBS-seq and other 5hmC detection methods for cfDNA applications (Li et al., 2024).

Aging and Epigenetic Clocks

5hmC levels change with age in a tissue-specific manner, and these changes are distinct from age-associated 5mC alterations. oxBS-seq enables construction of 5hmC-specific epigenetic clocks and identification of age-associated DhMRs that may contribute to functional decline. Human blood cell 5hmC has been shown to decline steadily with age.

Environmental Exposure and Population Epigenetics

Environmental factors including smoking, diet, and chemical exposures can alter the DNA hydroxymethylation landscape. A 2024 EWAS using BS+oxBS treatment distinguished smoking-associated 5hmC changes from 5mC changes at single-CpG resolution (Biomolecules, 2024), demonstrating the added value of oxBS over standard BS-seq for population epigenetics studies.

Case

Case Study: 5hmC at Cancer Mutation Hot Spots for Early Detection

5hmC Enrichment at Cancer Mutation Hot Spots

Source: Lu MJ & Lu Y, BMC Research Notes, 2022. https://doi.org/10.1186/s13104-022-06028-w

Background. Universal non-invasive genomic screening for early cancer detection requires biomarkers that are both cancer-specific and detectable in circulating cell-free DNA. While mutation-based screening has dominated the field, 5hmC emerged as a stable epigenetic modification with distinct tissue-specific patterns that could complement or improve upon mutation-based approaches.

Methods. The study applied oxidative bisulfite sequencing to map 5hmC modifications at or near known cancer mutation hot spots across multiple cancer types. Paired BS-seq and oxBS-seq libraries enabled single-base resolution discrimination of 5hmC from 5mC at each interrogated locus.

Results. 5hmC modifications were found to cluster at or near cancer mutation hot spots, and the patterns were cancer-type-specific — distinguishable from normal tissue 5hmC profiles. The distinct 5hmC signatures at these clinically relevant genomic positions suggest that 5hmC profiling could provide complementary biomarker information beyond mutation detection alone.

Figure from Lu & Lu, BMC Research Notes, 2022 — 5hmC enrichment at or near cancer mutation hot spots across multiple cancer types, supporting 5hmC as a potential target for early cancer detection.

Conclusion. This study demonstrates that oxBS-seq can identify cancer-relevant 5hmC signatures at mutation hot spots with single-base resolution. The findings support 5hmC profiling as a complementary approach to mutation-based cancer screening, with potential future applications in cfDNA-based liquid biopsy for early detection.

Method Comparison

Method Comparison: oxBS-seq vs Alternative 5hmC Detection Approaches

For researchers comparing technologies, the table below situates oxBS-seq among the main 5hmC detection methods.

Feature	oxBS-seq	TAB-seq	ACE-seq
Principle	Chemical oxidation (KRuO₄) + bisulfite	Enzymatic protection (β-GT) + TET oxidation + bisulfite	Enzymatic deamination (APOBEC3A), bisulfite-free
5hmC detection	Indirect (BS − oxBS subtraction)	Direct (reads as C)	Direct (resistant to deamination)
Resolution	Single-base	Single-base	Single-base
DNA degradation	Moderate (bisulfite step)	Higher (bisulfite + multi-step enzymatic)	Minimal (enzymatic only, no bisulfite)
Input requirement	Moderate to high	Moderate to high	Low (gentle enzymatic workflow)
Well validated	Yes — gold standard since 2012	Yes — published 2012	Emerging — bisulfite-free alternative
Best for	Genome-wide quantitative 5mC + 5hmC at single-base resolution	Direct 5hmC detection without subtraction	Low-input samples; FFPE; DNA integrity preservation is critical

oxBS-seq remains the most widely published and validated method for single-base resolution 5hmC quantification. Its paired-library design provides both 5mC and 5hmC data from the same sample — a unique advantage when both marks are biologically relevant.

FAQ

Frequently Asked Questions

Q: What is the difference between oxBS-seq and standard bisulfite sequencing (BS-seq)?

A: Standard BS-seq cannot distinguish 5-methylcytosine (5mC) from 5-hydroxymethylcytosine (5hmC) — both resist bisulfite conversion and are read as cytosine. oxBS-seq adds a selective chemical oxidation step (KRuO₄) that converts 5hmC to 5-formylcytosine before bisulfite treatment. The oxidized 5fC is then converted to uracil and read as thymine, while 5mC remains protected. By comparing a standard BS library with an oxBS library from the same DNA sample, true 5hmC levels are derived at each CpG site.

Q: Why do I need paired BS and oxBS libraries for each sample?

Q: What is the difference between oxWGBS, oxRRBS, and Targeted oxBS?

A: All three methods provide single-base resolution 5hmC quantification using the same oxidation chemistry. The difference is genomic scope: oxWGBS covers the entire genome; oxRRBS enriches for CpG-dense regions (promoters, CpG islands) via MspI digestion; Targeted oxBS focuses on user-specified genomic loci via capture probes or amplicons. Choose based on your discovery scope and sample budget — oxWGBS for comprehensive maps, oxRRBS for cost-effective regulatory region screening, Targeted oxBS for focused validation.

Q: How much DNA do I need for oxBS-seq?

Q: Does oxBS-seq work with FFPE or degraded DNA?

A: FFPE-derived DNA introduces additional challenges — formalin fixation causes crosslinks and fragmentation that reduce available intact template. However, the oxidative bisulfite chemistry is gentler on DNA than standard bisulfite alone, and FFPE samples are accepted on a project evaluation basis. Higher input amounts are typically needed to compensate for DNA degradation. We recommend submitting a DV200 trace if available, and we can assess feasibility for your specific samples.

Q: How is 5hmC quantified from paired BS and oxBS data?

A: For each CpG site with sufficient coverage in both libraries, the 5hmC level is calculated as: 5hmC = methylation ratio (BS library) − methylation ratio (oxBS library). The BS library reports the fraction of reads with C at the site (5mC + 5hmC). The oxBS library reports the fraction of reads with C (5mC only, since 5hmC was oxidized and reads as T). The subtraction yields the 5hmC fraction. Statistical confidence depends on sequencing depth at each CpG — deeper coverage yields narrower confidence intervals for the 5hmC estimate.

Q: What bioinformatics tools are used for oxBS-seq data analysis?

A: Our pipeline uses established tools for bisulfite-aware alignment (Bismark), methylation calling, and differential analysis (methylKit, DSS). For projects requiring refined oxi-mC modeling that accounts for incomplete oxidation or conversion, probabilistic deconvolution methods can be applied. Standard deliverables include per-site methylation calls from both libraries, 5hmC estimates, differential analysis results, and publication-ready visualizations. See our Epigenomic Data Analysis page for additional detail.

Q: Can oxBS-seq distinguish 5hmC from other oxidized 5mC derivatives like 5-formylcytosine (5fC)?

Q: Is oxBS-seq compatible with non-model organisms?

References

Booth MJ, Branco MR, Ficz G, et al. Quantitative Sequencing of 5-Methylcytosine and 5-Hydroxymethylcytosine at Single-Base Resolution. Science, 2012, 336:934-937. https://doi.org/10.1126/science.1220671
Booth MJ, Ost TWB, Beraldi D, et al. Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine. Nature Protocols, 2013, 8:1841-1851. https://doi.org/10.1038/nprot.2013.115
Lu MJ, Lu Y. 5-Hydroxymethylcytosine (5hmC) at or near cancer mutation hot spots as potential targets for early cancer detection. BMC Research Notes, 2022, 15:362. https://doi.org/10.1186/s13104-022-06028-w
Li JJN, Liu G, Lok BH. Cell-Free DNA Hydroxymethylation in Cancer: Current and Emerging Detection Methods and Clinical Applications. Genes, 2024, 15(9):1160. https://doi.org/10.3390/genes15091160
Rech GE, Lau AC, Goldfeder RL, et al. Global DNA Methylomes Reveal Oncogenic-Associated 5hmC Signatures in cfDNA of Cancer Patients. medRxiv, 2025. https://doi.org/10.1101/2025.01.09.25320283

oxBS-seq Services for DNA Hydroxymethylation Research