Methylation Sequencing: Principle, Methods, Steps, Uses microbiologystudy

Methylation sequencing is a method of sequencing used to study DNA methylation patterns across the genome which is an important biological process that adds methyl group (CH₃) to the cytosine bases of the DNA molecule.

DNA methylation is involved in different biological processes such as development, aging, cellular proliferation, and differentiation and also contributes to different diseases. Understanding DNA methylation patterns can help study how methylation influences gene expression, cellular functions, and disease mechanisms. Due to its role in different biological processes and diseases, it is important to study DNA methylation. Methylation sequencing allows high-resolution mapping of methylated cytosines across the genome. 

What is DNA Methylation?

DNA methylation is an important epigenetic modification that usually occurs at the C-5 position of cytosine bases in cytosine-phospho-guanine (CpG) dinucleotides.

• CpG dinucleotides cluster in regions called CpG islands which contain high GC content and are found at gene regulatory sites such as promoter regions.  
• DNA methyltransferases (DNMTs) are enzymes that add methyl groups to cytosine bases to form 5-methylcytosine (5-mC).
• On the other hand, demethylation can occur during replication or actively through enzymes like TET (ten-eleven translocase) which convert 5-mC to 5-hydroxymethylcytosine (5-hmC).
• Although 5-hmC is much less common than 5-mC, it plays an important role in regulating gene activity, particularly in regions like transcription start sites and regulatory regions.
• Cytosine methylation plays an important role in controlling gene expression. Methylation of promoter regions can lead to inactivation of gene activity. Methylation can block transcription factors from binding to DNA. Methylated CpGs recruit specific proteins such as methyl-CpG binding domain (MBD) which bring in repressive complexes that silence transcription.  
• There are different methods used to measure DNA methylation such as using restriction enzymes, affinity enrichment, or bisulfite conversion. The treated DNA can be analyzed using tools like microarrays and sequencing. Among these methods, genome-wide bisulfite sequencing is considered the most accurate and reliable for studying methylation. 
• Recent advancements in next-generation sequencing (NGS) and single-cell methylation sequencing have improved the ability to detect methylation changes with higher sensitivity and resolution and allow analysis at the single-cell level.

Principle of Methylation Sequencing

Methylation sequencing is based on detecting methylated cytosines in the DNA sequence. The most common method to do this is bisulfite conversion, where DNA is treated with sodium bisulfite. This chemical converts unmethylated cytosine to uracil. After PCR amplification, the uracils are read as thymines. The DNA is then prepared for sequencing. After sequencing, bioinformatics tools are used to analyze the data. These tools check the quality of the sequences, align them to the genome, and identify which cytosines are methylated. This allows differentiation between methylated and unmethylated cytosines by comparing the treated DNA sequence with the reference DNA sequence.

Methods of Methylation Sequencing

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

A. Restriction enzyme-based methods

• These methods use enzymes that cut only unmethylated DNA sequences. The DNA fragments are sequenced to identify which regions are not methylated. 
• MRE-seq (Methylation-Sensitive Restriction Enzyme Sequencing) involves digestion with methylation-sensitive restriction enzymes (MREs) followed by sequencing of the fragments to detect methylation. MREs like MspI and HpaII are often used. 

B. Affinity enrichment-based methods

• These methods use proteins or antibodies that bind specifically to methylated DNA. 
• MeDIP-seq (Methylated DNA Immunoprecipitation Sequencing) uses antibodies that bind to 5-mC to capture methylated DNA fragments. These fragments are then sequenced to identify methylation patterns.
• MBD-seq (Methyl-CpG Binding Domain Sequencing) uses MBD proteins to selectively bind to methylated regions of the genome which are then sequenced.

C. Bisulfite conversion-based methods

This is the most widely used method that converts unmethylated cytosines to uracil by using sodium bisulfite. When sequenced, the uracil is read as thymine. This helps to distinguish methylated from unmethylated regions. Different sequencing methods use bisulfite conversion.

Whole Genome Bisulfite Sequencing (WGBS)

• This method sequences the entire genome after bisulfite treatment and provides a detailed view of methylation patterns across the genome.
• It gives a comprehensive view of methylation across all CpG sites in the genome.

Reduced Representation Bisulfite Sequencing (RRBS)

• It focuses on CpG-dense regions of the genome and enriches CpG islands followed by bisulfite conversion and sequencing. 
• In this method, only specific regions of the genome are sequenced which reduces costs and complexity.

Targeted Bisulfite Sequencing

• It includes techniques like amplicon methyl-seq or target enrichment where specific genomic regions of interest are sequenced for methylation analysis.
• It focuses on specific genomic regions such as gene promoters using capture methods combined with bisulfite sequencing. 

There are other advanced methods of sequencing besides the above methods.

DNA hydroxymethylation Sequencing

• Oxidative Bisulfite Sequencing (OxBS-Seq) and Tet-Assisted Bisulfite Sequencing (TAB-Seq) are variants of bisulfite sequencing that focus on distinguishing between cytosine modifications such as 5-mC and 5-hmC. 
• OxBS-seq involves a selective oxidative step where 5-hmC is oxidized to 5-formyl cytosine (5-fC) which is deaminated to uracil and read as thymine during sequencing. 5-mC and 5-hmC can be distinguished by comparing the results of OxBS-seq with conventional bisulfite sequencing.
• TAB-seq allows direct detection of 5hmC. In this method, 5hmC is protected by adding a glucose molecule preventing its oxidation. It is less damaging to DNA compared to OxBS-seq but requires highly active Tet proteins for efficient conversion. It is more sensitive in detecting hydroxymethylation.

Single-Cell Bisulfite Sequencing (scBS-Seq)

• This method sequences methylation patterns at the level of individual cells.
• Traditional bisulfite sequencing methods require large amounts of DNA which limits their use to bulk cell populations. scBS-seq overcomes this by reducing the input requirement to a single cell. 
• One key innovation in scBS-seq is Post Bisulfite Adapter Tagging (PBAT) which involves ligating adapters after bisulfite treatment. This minimizes the amount of DNA needed.

Third-Generation Sequencing Methods

• Nanopore sequencing and SMRT sequencing can also be used in methylation sequencing. 
• These methods do not require bisulfite conversion or enrichment steps. They directly detect DNA methylation by reading the native DNA molecule.

Process/Steps of Methylation Sequencing

There are several methylation sequencing methods with different protocols. The methylation-specific DNA pre-treatment steps differ in each method. Since most methods use bisulfite conversion, the general workflow of methods involving bisulfite conversion is given below.

1. DNA Extraction: At first, high-quality genomic DNA is extracted from different samples of interest using different protocols and DNA extraction kits. For single-cell methods, isolation of individual cells is required before extraction.
2. Library Preparation: After DNA extraction, the DNA is fragmented into smaller pieces using either mechanical or enzymatic methods. This step involves end repair and adapter ligation. Some methods ligate adapters before bisulfite conversion, while others (e.g., PBAT) ligate adapters after bisulfite conversion to prevent DNA degradation. The fragments are then size-selected to ensure uniformity in sequencing. This is usually done using gel electrophoresis or magnetic beads to select DNA fragments of a desired size range for effective sequencing.
3. Bisulfite conversion: In this method, unmethylated cytosines are chemically converted into uracils which are then amplified as thymines during PCR. At first, DNA is denatured into single strands. Then sodium bisulfite is applied which causes unmethylated cytosines to deaminate and convert to uracil.
4. PCR Amplification: After bisulfite conversion, the DNA fragments are amplified by PCR. During this amplification, the uracils are replaced by thymines. This step increases the quantity of DNA to a level that can be sequenced.
5. Sequencing: The prepared library is sequenced using next-generation sequencing (NGS) platforms which read the bisulfite-treated DNA and generate data on cytosine methylation patterns.
6. Data Analysis: Data analysis includes data processing, quality control, data visualization, and interpretation. The sequencing reads are first checked for quality. Low-quality reads are discarded and adapters are trimmed to remove adapter contamination. The cleaned reads are aligned to a reference genome and methylation calls are made by comparing the treated DNA sequence to the original. The resulting methylation data is interpreted to identify methylation patterns. DNA methylation patterns are visualized and post-alignment processing is done to filter high-quality CpGs and trim overlapping paired-end reads. Methylation data can be visualized using genome browsers like Ensembl. 

Advantages and Limitations of Methylation Sequencing Methods

Methods	Advantages	Limitations
Restriction enzyme-based methods	It is suitable for targeted analysis of specific regions.It is cost-effective and easy to perform.	Restriction sites may not cover important methylation regions.It has lower coverage as it relies on specific enzyme sites.
Affinity-based methods	It can handle low-input samples.It enriches methylated DNA in CpG-rich regions.	It does not provide single-base resolution.It is biased towards regions with high methylation density.
WGBS	It has high resolution and provides genome-wide methylation data.	It requires large amounts of input DNA. It is expensive and computationally intensive. Bisulfite treatment can damage DNA.
RRBS	It is cost-effective and requires less DNA than WGBS. It focuses on CpG-rich regions.	This method is limited to CpG-dense regions limiting the scope of the analysis.It is less suitable for non-mammalian genomes.
Targeted bisulfite sequencing	It focuses on specific regions of interest. It is cost-effective and faster.	It misses genome-wide information and provides only a partial view of the methylome.
OxBS-Seq	It distinguishes between 5-mC and 5-hmC.	It has a more complex workflow than standard bisulfite methods. It is costly due to additional steps.
TAB-Seq	It specifically detects 5hmC.It has a high resolution for hydroxymethylation mapping.	It is expensive and has a complex protocol with additional oxidation steps.
scBS-Seq	It allows methylation analysis at the single-cell level.	It has lower coverage compared to bulk methods.

Applications of Methylation Sequencing

• Methylation sequencing allows quantification of DNA methylation and helps us better understand how DNA methylation influences cellular processes.
• Methylation sequencing is useful in identifying age-associated epigenetic markers. It provides valuable information about the aging mechanism and how epigenetic changes contribute to age-related conditions like neurodegenerative diseases and cardiovascular issues.
• Abnormal DNA methylation patterns are often linked to disruptions in normal body functions and genomic instability which can contribute to diseases such as cancer. Methylation sequencing can identify tumor-specific methylation patterns which can be used as biomarkers to detect cancer early and tailor treatments to individual patients.
• External factors like diet, pollution, and stress can influence DNA methylation. Methylation sequencing can be used to study how environmental factors modify the genome and contribute to diseases.
• Methylation sequencing can help to understand the functional mechanisms of complex diseases.

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