oxBS-seq Services for DNA Hydroxymethylation Research

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.

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.

1. Sample Receipt and Quality Assessment — QC Checkpoint: DNA quantity (Qubit) and purity (OD260/280); genomic integrity by gel electrophoresis or Bioanalyzer/TapeStation; concentration verification.
2. 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.
3. 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.
4. Data QC — QC Checkpoint: Alignment rates for both BS and oxBS libraries; coverage correlation between paired libraries; per-sample methylation summary statistics; replicate correlation.
5. Bioinformatics and Report Delivery — QC Checkpoint: 5hmC call reproducibility; differential analysis statistical metrics; deliverable completeness verification; final QC summary.

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:

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

Applications Across Research Areas

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.

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.