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DNA Methylation

Unlocking the Secrets of Epigenetics

Assess the DNA methylation profile of your samples with Epistem's expertise in Next Generation Sequencing (NGS). Whether you're working with in vitro, pre-clinical, or patient samples, our services can provide valuable insights. Choose between reduced representation bisulfite sequencing (RRBS) or whole genome bisulfite sequencing (WGBS) based on your project requirements.

What's Available

Uncover DNA methylation insights with our streamlined RRBS and WGBS workflows.

Reduced Representation Bisulfite Sequencing

Unlock DNA Methylation Insights with RRBS: A powerful NGS-based sequencing method that combines bisulfite sequencing with restriction enzymes. It enables the assessment of DNA methylation status in high CpG content regions. The specific restriction digestion used in this technique targets CpG motifs, providing comprehensive coverage of transcriptionally active components, including promoters, enhancers, CpG islands, gene bodies, repetitive DNA sequences, and regulatory elements.

RRBS serves as an excellent platform for clinical applications, such as pilot studies and biomarker discovery, allowing for comparative analysis of DNA methylation levels between different treatment groups, such as treated versus control tissue.

 

Project Workflow

1. DNA Extraction and Quality Assessment: We extract and evaluate the quality and quantity of the DNA of interest.

2. Tailored DNA Fragmentation: Using a restriction enzyme digest, we create DNA fragments of specific lengths. MspI, the most common enzyme in RRBS, is used for this purpose.

3. Repair and Adapter Ligation: We repair the 5′ CG overhangs and add A-tails to facilitate efficient ligation with Illumina adapters, which have a 3′-T overhang.

4. Size Selection: The DNA fragments are carefully chosen in the range of approximately 40-120 bp and 120-220 bp to optimize sequencing performance.

5. Bisulfite Conversion: Non-methylated cytosines are deaminated, converting them to uracils that are subsequently read as thymidines during sequencing. Methylated cytosines remain protected and are read as cytosines.

6. PCR Amplification and Indexing: The bisulfite-converted DNA is amplified through PCR, and unique indexes are added to enable sample identification and multiplexing.

7. Illumina NGS Sequencing: The indexed samples are then sequenced on our advanced Illumina NGS platforms, generating high-quality and high-throughput data.

8. Bioinformatic Analysis: The final step in the RRBS protocol involves comprehensive bioinformatic analysis, including sequence alignment and identification of methylation patterns.

Our streamlined RRBS workflow ensures accurate and efficient analysis of DNA methylation. Join us to unlock the secrets hidden within your samples.

Whole Genome Bisulfite Sequencing

WGBS is a cutting-edge technique that combines bisulfite treatment with NGS, enabling comprehensive assessment of the methylome of any organism at a single-base resolution. This gold standard method, endorsed by the International Human Epigenome Consortium, provides complete genome-wide coverage of all CpGs, offering unparalleled epigenetic profiling.

With WGBS, we can accurately analyze the methylation status of every CpG, CHG, and CHH locus in your sample of interest. This empowers us to identify differentially methylated loci between various types, such as healthy control patients and those diagnosed with cancer.

By profiling the entire methylome, WGBS opens doors to study developmental programming, cell differentiation, cell cycle and DNA repair mechanisms, as well as genomic imprinting. It offers a comprehensive understanding of the intricate epigenetic landscape.

 

Project Workflow

1. DNA Extraction and Quality Assessment: We begin by extracting the DNA of interest and conducting rigorous quality and quantity evaluations.

2. Bisulfite Conversion: The extracted DNA undergoes bisulfite conversion, which enables the distinction between methylated and unmethylated cytosines.

3. Random-Priming and Unique Tagging: Single-stranded DNA is randomly primed using a polymerase capable of reading uracil nucleotides. This process synthesizes DNA strands that incorporate a unique sequence tag.

4. Selective Labeling and Adapter Addition: The 3′ end of the DNA strand is selectively labeled with a second sequence. Next, Illumina index adapters are added to both the 5′ and 3′ ends using PCR amplification.

5. Illumina NGS Sequencing: The PCR product is ready for sequencing on our state-of-the-art Illumina NGS platforms, ensuring high-quality and high-throughput data generation.

6. Bioinformatics Analysis: Our comprehensive bioinformatics pipeline includes the assessment of methylation density, identification of differentially methylated regions (DMRs), annotation and enrichment analysis (GO/KEGG), and clustering analysis.

Unleash the power of our streamlined workflow to explore DNA methylation patterns. Join us in unlocking the epigenetic secrets hidden within your samples.

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