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5mC/5hmC Sequencing

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DNA methylation and hydroxymethylation at the 5-position of cytosine (5mC) are essential epigenetic alterations that play vital roles in gene expression regulation and cell differentiation. DNA methylation typically represses gene transcription. Hydroxymethylation, on the contrary, activates gene expression or prompts DNA demethylation. 5mC groups introduced by DNA Methyl-Transferases can be iteratively oxidized to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) by the action of Tet Methylcytosine Dioxygenases. 5hmC is the most abundant component in vivo among the three 5mC oxidative derivatives and can be identified in almost all mammalian tissues and cells. 5mC and 5hmC genome-wide mapping uncover the genomic areas of these alterations, which is crucial for elucidating their mechanisms.

5mC/5hmC: the cytosine methylation cycleFigure 1. The cytosine methylation cycle (Ecsedi, 2018)

CD Genomics is a biotechnology company focusing on sequencing technology. With cutting-edge sequencing technology and in-depth bioinformatics analysis, we assist customers in mining genomics information. Our areas of expertise include genomics, transcriptomics, epigenetics, population genetics, microbiology, and many other fields. Our high-throughput 5mC/5hmC sequencing service utilizes multiple mature and stable platforms with high efficiency, simplicity, and accuracy to help your epigenetics research.

CD Genomics can provide genome-wide 5mC and 5hmC detection by following approaches:

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

2. Oxidative bisulfite sequencing (oxBS-seq)

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

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

5mC/5hmC Sequencing Workflow

Our long-standing experience and advanced platforms allow us to provide a comprehensive service package from project consultation, experiment design, sequencing, to bioinformatics analysis. We strive to provide the most suitable scheme for your project.

5mC/5hmC Sequencing Workflow

Service Specifications

Sample Requirements Sample Requirements

  • Starting amount of total DNA ≥ 5 µg(WGBS); ≥ 3 µg(RRBS, hMeDIP)
  • Sample concentration ≥ 50 ng/µl
  • All DNA samples are validated for purity and quantity
Sequencing Strategy

  • Illumina, PE150
  • More than 80% of bases with a ≥Q30 quality score
Bioinformatics Analysis Bioinformatics Analysis
We provide custom bioinformatics analysis services:

  • Sequencing Data Quality Control
  • Alignment against reference genomes
  • 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
  • Immunoenriched fragment analysis
  • Peak analysis
  • Motif analysis

References:
1. Ecsedi S, Rodríguez-Aguilera JR, Hernandez-Vargas H. 5-Hydroxymethylcytosine (5hmC), or how to identify your favorite cell. Epigenomes. 2018, 2(1):3.
2. Kirschner K, Krueger F, Green AR, Chandra T. Multiplexing for Oxidative Bisulfite Sequencing (oxBS-seq). InDNA Methylation Protocols 2018. Humana Press.

* For Research Use Only. Not for use in diagnostic procedures.
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