Methylation Sequencing (5mC and 5hmC)
What Is Methylation Sequencing (5mC and 5hmC)?
Enzymatic Methylation Sequencing (EM-Seq) is an advanced next-generation sequencing (NGS) method for accurate detection of DNA methylation—specifically 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC)—across the genome. Unlike traditional whole genome bisulfite sequencing (WGBS), which chemically converts unmethylated cytosines and causes significant DNA degradation, EM-Seq uses enzymatic conversion to protect DNA integrity and deliver a truer representation of the methylome.
The EM-Seq workflow, developed by New England Biolabs (NEB), uses two key enzymatic steps:
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TET2 enzyme converts 5mC to 5-carboxylcytosine (5caC), which is resistant to deamination.
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Oxidation Enhancer modifies 5hmC to 5-glycosylhydroxymethylcytosine (5ghmC), also resistant to deamination.
Subsequently, APOBEC selectively deaminates unmodified cytosines to uracils, which are read as thymines during sequencing. In this system:
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Unmethylated cytosines are sequenced as thymines.
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5mC and 5hmC remain as cytosines, preserving their original identity.
The result is a highly accurate methylation profile with minimal DNA fragmentation, reduced GC bias, and superior CpG detection compared to bisulfite methods. AUGenomics leverages this technology to provide comprehensive 5mC and 5hmC analysis with lower input requirements, making it ideal for challenging samples such as cfDNA and FFPE DNA.
Advantages of Methylation Sequencing (5mC and 5hmC)
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Non-Bisulfite Method: Avoids harsh chemical treatment, preserving DNA integrity.
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High Accuracy: Distinguishes between 5mC and 5hmC with precise base-resolution detection.
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Lower Input Requirements: Works with as little as 10 ng of DNA, enabling use with cfDNA and degraded samples.
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Reduced DNA Damage: Enzymatic conversion prevents fragmentation, resulting in longer insert sizes and more complex libraries.
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Superior CpG Coverage: Detects more CpGs at greater depth compared to WGBS, improving confidence in methylation calls.
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Minimal GC Bias: Provides even coverage across GC- and AT-rich regions, reducing sequencing artifacts.
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Cost-Effective: More CpGs are detected per read, reducing sequencing costs per usable data point.
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Broad Sample Compatibility: Suitable for genomic DNA, cfDNA, FFPE DNA, and low-input samples.
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Advanced Bioinformatics: Delivers methylation maps, 5mC/5hmC quantification, and differential methylation analysis.
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Expert Consultation: AUGenomics guides you through sample preparation, data analysis, and biological interpretation.
EM-seq is a powerful tool to study DNA methylation dynamics in health and disease, particularly in tissues where 5hmC plays critical roles, such as the brain, cancer, and stem cells. Common applications include:
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Cancer Epigenetics: Detecting methylation changes in tumor suppressor genes or oncogenes.
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Neurological Disorders: Investigating methylation and hydroxymethylation patterns associated with brain development and disease.
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Developmental Biology: Mapping methylation dynamics during cell differentiation and embryogenesis.
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Autoimmune and Inflammatory Diseases: Profiling epigenetic modifications involved in immune dysregulation.
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Biomarker Discovery: Identifying epigenetic markers for diagnostics or therapeutic targeting.
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cfDNA Analysis: Leveraging low-input capability for liquid biopsy studies in cancer detection and monitoring.
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FFPE Sample Analysis: Unlocking methylation data from archived tissue samples.
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Translational Medicine: Enabling methylation studies that support personalized medicine approaches.
What is Methylation Sequencing (5mC and 5hmC) Used For?
Methylation Sequencing (5mC and 5hmC)with AUGenomics
Sample Submission
We accept high-quality genomic DNA or nuclei preparations for EM-seq. For best results, DNA should be non-degraded and free of contaminants. Contact us for advice on low-input or difficult samples.
Please refer to our Shipping Guidelines for project-specific guidance.
Turnaround Time
Typical turnaround for EM-seq projects is 10–14 business days from sample receipt. Expedited options are available depending on project scope and sequencing depth.
Frequently Asked Questions (FAQs)
Q: Is EM-seq suitable for cell-free DNA or FFPE samples?
A: Yes, although results may vary based on DNA quality. Contact us to discuss project feasibility for degraded or fragmented samples.
Q: How does EM-Seq compare to bisulfite sequencing (WGBS)?
A: EM-Seq avoids DNA degradation and biases associated with bisulfite treatment, resulting in higher library complexity, more uniform coverage, and greater CpG detection with the same sequencing depth.
Q: Can EM-Seq distinguish between 5mC and 5hmC?
A: Yes. Unlike bisulfite sequencing, EM-Seq’s enzymatic workflow accurately differentiates 5mC and 5hmC from unmethylated cytosines.
Q: What is the minimum DNA input required?
A: AUGenomics can generate high-quality data from as little as 10 ng of DNA, making this method ideal for low-input samples.
Q: What sequencing depth is recommended?
A: Recommended depth varies by application (e.g., whole genome vs. targeted regions). Our team will recommend an optimal strategy based on your research goals.
Q: Can EM-Seq data be integrated with other analyses?
A: Yes. EM-Seq data can be combined with transcriptomics, chromatin accessibility (ATAC-seq), or other omics datasets for multi-dimensional epigenetic insights.
Got more questions? Contact our team and get a free consultation anytime. info@augenomics.com
Glossary of Terms
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5mC: 5-methylcytosine, a DNA modification associated with transcriptional repression
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5hmC: 5-hydroxymethylcytosine, an oxidized form of 5mC with distinct regulatory functions
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Bisulfite sequencing: Traditional method for detecting DNA methylation by converting unmethylated cytosines
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EM-seq: An enzymatic approach to DNA methylation profiling that avoids bisulfite-induced damage
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Epigenetics: Study of heritable changes in gene expression without alterations in DNA sequence
