DNA methylation and hydroxymethylation are important epigenetic modifications for gene expression regulation. DNA methylation typically represses gene transcription. Hydroxymethylation, on the contrary, activates gene expression or prompts DNA demethylation.

5-Hydroxymethylcytosine (5hmC) is converted from 5-methylcytosine (5mC) by a group of enzymes termed ten-eleven translocation (TET) family dioxygenases. The gold-standard bisulfite conversion technologies to study DNA methylation do not distinguish between 5mC and 5hmC. However, new approaches to mapping 5hmC genomewide have advanced rapidly, although it is unclear how the different methods compare in accurately calling 5hmC.

CD Genomics can provide three 5hmC genome-wide detection approaches:

1. Whole-genome bisulfite/oxidative bisulfite sequencing (WGBS/WGoxBS-seq)

2. Reduced representative bisulfite oxidative bisulfite sequencing (RRBS/RRoxBS)

3. Antibody-based immunoprecipitation and sequencing of hydroxymethylated DNA (hMeDIP-seq).

4. Targeted DNA bisulfite/hydroxymethylation sequencing (TBS/ oxTBS-seq)

5. 5hmC selective chemical labelling technology (5hmC -Seal)

The Introduction of oxBS-Seq

5-methyl cytosine (5mC) can be oxidised by a group of oxigenases called ten-eleven translocation enzymes (Tets) . Under consumption of oxygen and 2-oxoglutarate, these Fe(II) dependent dioxigenases oxidise 5mC in a first reaction to 5-hydroxymethyl cytosine (5hmC), followed by 5-formyl cytosine (5fC) and finally 5-carboxy cytosine (5caC). The most abundant formof these oxidised cytosine variants is 5hmC.

Oxidative bisulfite conversion (oxBS), a pre-bisulfite oxidation reaction converts 5hmC to 5fC, which will be converted by bisulfite to 5f-uracil and to thymine in the subsequent PCR. This library is then compared to a traditional bisulfite sequencing library (BS-seq) constructed on the same sample to determine the amount of 5mC and 5hmC for each modified cytosine within the DNA. oxBS-seq has a relatively simple and fast experimental workflow and can obtain both the methylome and hydroxymethylome simultaneously.


oxBS-Seq Workflow

Genomic DNA was processed to achieve 10 kb fragments. Fragmented DNA was concentrated using purification columns. This fragmented and purified DNA was taken forward for oxidation + bisulfite treatment. Buffer exchange, denaturation, oxidation, bisulfite conversion, cleanup, and qualitative assessment of 5-hydroxymethylcytosine oxidation was carried out. Last, oxBS- and BS-converted DNA were used to construct oxBS-seq and corresponding BS-seq libraries. All double and single stranded DNA was quantified on an Agilent 2100 Bioanalyser before cluster generation and sequencing on illumina platform according to the manufacturer’s protocols.


Service Specifications

Sample Requirements Sample Requirements:
  • Starting amount of Genomic DNA ≥ 4
  • µg(oxWGBS-Seq); ≥ 2 µg(OxRRBS-Seq); ≥ 1 µg(oxTBS); ≥ 3 µg(hMeDIP-seq); ≥ 3 µg( 5hmC Seal-seq)
  • Sample concentration ≥ 50 ng/µl
  • All DNA samples are validated for purity and quantity
  • For cfDNA,15-40ng( 5hmC Seal-seq)
Sequencing Strategy:
  • Illumina, PE150
  • WGBS+oxWGBS: >=30X
  • RRBS+oxRRBS: 10G data)
  • TBS+oxTBS: sequencing depth>=200X
  • hMeDIP-seq(IP+Input): >=20M reads
  • 5hmC Seal-seq(IP+Input): >=20M reads
  • More than 80% of bases with a ≥Q30 quality score
Bioinformatics Analysis Bioinformatics Analysis
We provide customized bioinformatics analysis including:
  • Sequencing Data Quality Control
  • Alignment against reference genome
  • Whole genome methylation and hydroxymethylation level distribution
  • Correlation analysis
  • PCA analysis
  • Differential methylation analysis and differential hydroxymethylation analysis
  • Functional annotation with GO/KEGG analysis
  • Differential expression level analysis of peak associated genes


[1] Giehr, Pascal, Kyriakopoulos, et al. Two are better than one: HPoxBS - hairpin oxidative bisulfite sequencing[J]. Nucleic acids research, 2018.
[2] Skvortsova K , Zotenko E , Luu P L , et al. Comprehensive evaluation of genome-wide 5-hydroxymethylcytosine profiling approaches in human DNA[J]. Epigenetics & Chromatin, 2017, 10(1):16.

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