DNA Methylation Sequencing Methods

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Introduction

Bisulfite Sequencing: The gold standard for DNA methylation studies, converting unmethylated cytosines to uracil.
Enzymatic Methyl-seq (EM-seq): A newer method using enzymes to convert unmethylated cytosines, reducing DNA degradation.
Whole-Genome Bisulfite Sequencing (WGBS): Provides comprehensive single-base resolution DNA methylation maps.
Reduced Representation Bisulfite Sequencing (RRBS): Focuses on CpG-rich regions, reducing sequencing costs.
Methylated DNA Immunoprecipitation Sequencing (MeDIP-seq): Uses antibodies to capture methylated DNA, followed by sequencing.
Affinity Enrichment-Based Methods: Capture methylated DNA using methyl-binding proteins or antibodies.
Restriction Enzyme-Based Methods: Use enzymes that cut DNA at specific methylation sites.
Comparison of Methods: NEBNext Enzymatic Methyl-seq and Swift Accel-NGS Methyl-Seq kits perform well, especially with low DNA input.

Gold Standard: Bisulfite sequencing is considered the gold standard for DNA methylation studies.
Conversion Process: Converts unmethylated cytosines to uracil, which are then read as thymine during sequencing.
Applications: Used for genome-wide and targeted DNA methylation analysis.
Challenges: Bisulfite treatment can degrade DNA, making it less suitable for low-quality samples.
Tools: Commonly used tools include the Bismark algorithm for mapping bisulfite-treated reads.

New Method: Uses enzymes to convert unmethylated cytosines, reducing DNA degradation.
Enzymes Involved: TET2 oxidizes methylated cytosines, and APOBEC3A converts unmethylated cytosines to uracils.
Advantages: Less harsh on DNA compared to bisulfite treatment, making it suitable for low-quality samples.
Performance: NEBNext Enzymatic Methyl-seq kit performs well with both high and low DNA input.
Applications: Suitable for whole-genome methylation sequencing and targeted methylation analysis.

Comprehensive Mapping: Provides single-base resolution DNA methylation maps across the entire genome.
First Applied: Successfully applied to Arabidopsis thaliana in 2008 and humans in 2009.
Library Preparation: Adapter-ligated library material undergoes bisulfite conversion.
Challenges: Bisulfite conversion can degrade DNA, leading to lower quantities and diversity of sequenceable material.
Applications: Used for detailed epigenetic studies and understanding methylation patterns in various organisms.

Cost-Effective: Focuses on CpG-rich regions, reducing sequencing costs.
Library Preparation: Involves digesting DNA with restriction enzymes and selecting fragments for bisulfite conversion.
Applications: Suitable for studies where genome-wide coverage is not necessary.
Advantages: Provides high-resolution methylation data for targeted regions.
Tools: MethylKit app can analyze sequencing data from RRBS.

Capture Method: Uses antibodies to capture methylated DNA, followed by sequencing.
Specificity: Can target specific methylation marks such as 5-methylcytosine (5mC) or 5-hydroxymethylcytosine (5hmC).
Applications: Suitable for studying specific methylation patterns and identifying differentially methylated regions.
Advantages: Does not require bisulfite conversion, preserving DNA integrity.
Tools: MeDIP-seq is a common method used for this approach.

Capture Method: Uses methyl-binding proteins or antibodies to capture methylated DNA.
Applications: Suitable for genome-wide methylation profiling and identifying differentially methylated regions.
Advantages: Does not require bisulfite conversion, preserving DNA integrity.
Challenges: May have lower resolution compared to bisulfite sequencing.
Tools: Commonly used in combination with next-generation sequencing platforms.

Principle: Use enzymes that cut DNA at specific methylation sites.
Applications: Suitable for locus-specific methylation analysis.
Advantages: Can be combined with other methods for more comprehensive analysis.
Challenges: Limited to regions with specific enzyme recognition sites.
Tools: Often used in combination with next-generation sequencing for detailed analysis.