Whole Genome Methylation Sequencing via Enzymatic Conversion (EM-seq): Protocol, Data Processing, and Analysis.

Area of Science:

• Epigenetics
• Genomics
• Molecular Biology

Background:

• Whole genome bisulfite sequencing (WGBS) has been the standard for DNA methylation analysis for 15 years.
• WGBS is prone to technical biases, leading to overestimated methylation values and artifacts in cytosine-rich regions.
• Bisulfite conversion is a damaging process for DNA.

Purpose of the Study:

• Introduce and evaluate Enzymatic Methylation sequencing (EM-seq) as a superior alternative to WGBS.
• Highlight the advantages of EM-seq for DNA methylation analysis.
• Demonstrate the bias-free and degradation-free nature of EM-seq.

Main Methods:

• EM-seq employs a two-step enzymatic conversion process using TET2, T4-BGT, and APOBEC.
• Cytosines are protected via oxidation and glucosylation.
• Unmodified cytosines are deaminated to uracil.

Main Results:

• EM-seq is degradation-free and bias-free, unlike WGBS.
• The technique requires low DNA input and yields high library output with longer reads.
• EM-seq demonstrates uniform genomic coverage, reduced duplication, and accurate methylation detection across more CpGs.
• No sequence-specific artifacts were observed with EM-seq.

Conclusions:

• EM-seq represents a significant advancement in DNA methylation analysis, replacing the damaging WGBS method.
• The technique offers improved accuracy, efficiency, and reliability for base-resolution methylation studies.
• EM-seq is a robust, bias-free method suitable for various genomic applications requiring precise methylation profiling.