Advanced DNA Methylation Sequencing: Targeted Bisulfite Techniques and CpG Analysis

Table of Contents

• Introduction
• What is DNA Methylation?
• Principle of Methylation Sequencing
• Methods of Methylation Sequencing
• A. Restriction enzyme-based methods
• B. Affinity enrichment-based methods
• C. Bisulfite conversion-based methods
• Process/Steps of Methylation Sequencing
• Advantages and Limitations of Methylation Sequencing Methods 

Introduction

Methylation sequencing is a powerful technique used to investigate DNA methylation patterns across the entire genome. DNA methylation, which involves the addition of a methyl group (CH3) to cytosine bases, plays a critical role in various biological processes, including development, aging, cellular proliferation, and differentiation. 

It also contributes to the onset of various diseases. By analyzing DNA methylation, researchers can gain insights into how these modifications affect gene expression, cellular functions, and disease mechanisms. Given its significance in numerous biological processes and disease pathways, studying DNA methylation is essential. Methylation sequencing provides high-resolution mapping of methylated cytosines throughout the genome.

What is DNA Methylation?

DNA methylation is a key epigenetic modification that typically occurs at the C-5 position of cytosine bases within cytosine-phospho-guanine (CpG) dinucleotides.

• CpG dinucleotides are often clustered in regions known as CpG islands, which are rich in GC content and commonly found at gene regulatory sites, such as promoter regions.
• DNA methyltransferases (DNMTs) are enzymes responsible for adding methyl groups to cytosine, producing 5-methylcytosine (5-mC).
• Conversely, demethylation can happen during DNA replication or actively via enzymes like TET (ten-eleven translocase), which convert 5-mC into 5-hydroxymethylcytosine (5-hmC). Although 5-hmC is less abundant than 5-mC, it plays a critical role in regulating gene activity, especially in areas like transcription start sites and regulatory regions.
• Cytosine methylation is crucial for regulating gene expression. Methylation of promoter regions can suppress gene activity by preventing transcription factors from binding to DNA. Methylated CpG sites can attract proteins like methyl-CpG binding domain (MBD) proteins, which recruit repressive complexes that silence transcription.
• Various methods are available to assess DNA methylation, including the use of restriction enzymes, affinity enrichment, or bisulfite conversion. The treated DNA can then be analyzed through techniques like microarrays or sequencing, with genome-wide bisulfite sequencing being the most accurate and reliable for methylation studies.
• Advances in next-generation sequencing (NGS) and single-cell methylation sequencing have significantly enhanced the ability to detect methylation changes with greater sensitivity and resolution, enabling analysis at the single-cell level.

Principle of Methylation Sequencing

Methylation sequencing works by detecting methylated cytosines within the DNA sequence. The most widely used technique for this is bisulfite conversion, where DNA is treated with sodium bisulfite. This process converts unmethylated cytosines into uracils, which are then read as thymines after PCR amplification. The DNA is subsequently prepared for sequencing, and bioinformatics tools are employed to analyze the resulting data. These tools assess sequence quality, align the sequences to the reference genome, and identify methylated cytosines. By comparing the bisulfite-treated DNA sequence to the reference sequence, methylated cytosines can be distinguished from unmethylated ones.

Methods of Methylation Sequencing

Methylation sequencing methods can be broadly classified into three main categories based on how they detect methylation:

A. Restriction enzyme-based methods

• These methods utilize enzymes that selectively cut unmethylated DNA sequences. Sequencing the resulting DNA fragments helps identify regions lacking methylation.
• MRE-seq (Methylation-Sensitive Restriction Enzyme Sequencing): This approach involves digesting DNA with methylation-sensitive restriction enzymes (MREs) like MspI and HpaII, followed by sequencing the fragments to detect methylation.

B. Affinity enrichment-based methods

• These methods employ proteins or antibodies that specifically bind to methylated DNA.
• MeDIP-seq (Methylated DNA Immunoprecipitation Sequencing): Antibodies that recognize 5-mC are used to capture methylated DNA fragments, which are then sequenced to map methylation patterns.
• MBD-seq (Methyl-CpG Binding Domain Sequencing): MBD proteins selectively bind to methylated regions of the genome, which are subsequently sequenced to reveal methylation profiles.

C. Bisulfite conversion-based methods

This widely used method involves converting unmethylated cytosines to uracil via sodium bisulfite treatment. Upon sequencing, the uracils are read as thymines, enabling distinction between methylated and unmethylated cytosines. Several sequencing techniques utilize bisulfite conversion:

• Whole Genome Bisulfite Sequencing (WGBS): This method sequences the entire genome after bisulfite conversion, providing a comprehensive view of methylation patterns across all CpG sites.
• Reduced Representation Bisulfite Sequencing (RRBS): By focusing on CpG-rich regions, RRBS enriches CpG islands, reducing sequencing costs and complexity while targeting specific areas for bisulfite conversion and sequencing.
• Targeted Bisulfite Sequencing: This approach, including methods like amplicon methyl-seq or target enrichment, sequences specific genomic regions of interest, such as gene promoters, for detailed methylation analysis.

Additional Advanced Sequencing Methods:

DNA hydroxymethylation Sequencing

• Oxidative Bisulfite Sequencing (OxBS-Seq): This method selectively oxidizes 5-hmC to 5-formylcytosine (5-fC), which is converted to uracil and read as thymine during sequencing. By comparing OxBS-seq data with conventional bisulfite sequencing, 5-mC and 5-hmC can be differentiated.
• Tet-Assisted Bisulfite Sequencing (TAB-Seq): This method detects 5-hmC by protecting it with a glucose molecule, preventing its oxidation. It requires highly active Tet proteins and is less damaging to DNA than OxBS-seq, offering increased sensitivity for hydroxymethylation detection.

Single-Cell Bisulfite Sequencing (scBS-Seq)

• This technique enables the sequencing of methylation patterns at the single-cell level. Traditional bisulfite sequencing requires large amounts of DNA, limiting it to bulk populations. 
• scBS-seq overcomes this by using Post Bisulfite Adapter Tagging (PBAT), which ligates adapters after bisulfite treatment, reducing the DNA input requirement to just a single cell.

Third-Generation Sequencing Methods

• Nanopore Sequencing and SMRT Sequencing: These methods directly detect DNA methylation without the need for bisulfite conversion or enrichment steps. They read the native DNA molecule, enabling real-time detection of methylation patterns.

Process/Steps of Methylation Sequencing

The process of methylation sequencing involves several key steps, particularly in methods that use bisulfite conversion. Below is a general workflow for bisulfite conversion-based methylation sequencing:

1. DNA Extraction:

The process begins with the extraction of high-quality genomic DNA from various samples using specific protocols and DNA extraction kits. For single-cell methods, individual cells must be isolated prior to DNA extraction.

2. Library Preparation:

After extraction, the genomic DNA is fragmented into smaller pieces, either mechanically or enzymatically. This step includes end repair and adapter ligation. In some methods, adapters are ligated before bisulfite conversion, while in others, like Post-Bisulfite Adapter Tagging (PBAT), adapters are ligated afterward to minimize DNA degradation. Fragment size selection is performed next, typically using gel electrophoresis or magnetic beads, to ensure that only fragments of the desired size range proceed for sequencing.

3. Bisulfite Conversion:

This is the core step of the workflow. Here, unmethylated cytosines are chemically converted to uracils. First, the DNA is denatured into single strands, after which sodium bisulfite is applied. This chemical reaction causes unmethylated cytosines to deaminate, converting them into uracils, which will later be read as thymines during sequencing.

4. PCR Amplification:

Following bisulfite conversion, the DNA fragments undergo PCR amplification. During this process, uracils are replaced by thymines, increasing the quantity of DNA for sequencing. This step ensures that there is sufficient material to analyze methylation patterns.

5. Sequencing:

The amplified DNA library is then sequenced using next-generation sequencing (NGS) platforms, which read the bisulfite-treated DNA. This step generates the raw data that contains information about methylation patterns by distinguishing between methylated and unmethylated cytosines.

6. Data Analysis:

After sequencing, the data is processed through several steps:

• Quality Control: Sequencing reads are evaluated for quality, with low-quality reads being discarded, and adapters are trimmed to remove any contamination.
• Alignment: The cleaned reads are aligned to a reference genome, and methylation calls are made by comparing the bisulfite-treated DNA to the reference sequence.
• Visualization & Interpretation: DNA methylation patterns are visualized, often using genome browsers like Ensembl. Post-alignment processing is done to filter high-quality CpG sites and remove overlapping paired-end reads, providing a detailed view of methylation across the genome.

Advantages and Limitations of Methylation Sequencing Methods

Applications of Methylation Sequencing

• Quantifying DNA methylation to gain insights into how it regulates cellular processes.
• Identifying age-related epigenetic markers, providing crucial information on aging mechanisms and the role of epigenetic changes in age-related conditions such as neurodegenerative diseases and cardiovascular disorders.
• Detecting abnormal methylation patterns linked to genomic instability and disease, especially cancer. Tumor-specific methylation signatures identified through sequencing can serve as biomarkers for early detection and personalized treatment strategies.
• Investigating how environmental factors like diet, pollution, and stress influence DNA methylation, contributing to disease development.
• Uncovering the functional mechanisms of complex diseases by analyzing methylation patterns.