What is Whole Genome Bisulfite Sequencing (WGBS)?

Whole Genome Bisulfite Sequencing (WGBS) is a high-resolution technique used to map DNA methylation across the entire genome at single-base resolution. It involves the treatment of DNA with sodium bisulfite, which converts unmethylated cytosines into uracils (read as thymine during sequencing), while leaving methylated cytosines unchanged.

By sequencing the bisulfite-treated DNA and comparing it to the reference genome, researchers can precisely detect 5-methylcytosine (5mC) patterns — an important epigenetic modification linked to gene regulation, development, and disease.

Overview

• Purpose: Profile DNA methylation genome-wide

• Input: High-quality genomic DNA

• Methylation Type Detected: Primarily 5-methylcytosine (5mC) in CpG, CHG, and CHH contexts

• Output: Methylation status of individual cytosines across the genome

• Applications: Epigenetic regulation, cancer research, developmental biology, imprinting disorders

How WGBS Works

1. DNA Extraction
   High-purity genomic DNA is isolated from cells or tissues.

2. Bisulfite Conversion
   DNA is treated with sodium bisulfite, which converts unmethylated cytosines to uracils, while methylated cytosines remain unchanged.

3. Library Preparation
   Bisulfite-converted DNA is fragmented, adapter-ligated, and PCR-amplified.

4. Sequencing
   Libraries are sequenced on high-throughput platforms (e.g., Illumina NovaSeq, HiSeq).

5. Data Analysis
   Specialized tools align the bisulfite reads and calculate methylation ratios at each cytosine.

Applications of Whole Genome Bisulfite Sequencing

• Genome-Wide DNA Methylation Profiling
  Understand epigenetic regulation across the whole genome.

• Cancer Epigenomics
  Identify tumor-specific methylation changes, epigenetic silencing of tumor suppressors, or activation of oncogenes.

• Developmental Biology
  Study dynamic methylation changes during embryogenesis, cell differentiation, or aging.

• Environmental Epigenetics
  Assess how toxins, diet, or stress affect methylation patterns.

• Imprinting and X-Inactivation
  Examine allele-specific methylation in imprinted genes and dosage compensation.

• Epigenetic Biomarker Discovery
  Discover novel diagnostic, prognostic, or therapeutic methylation signatures.

Advantages of WGBS

• Single-Base Resolution
  Precise detection of methylated and unmethylated cytosines.

• Genome-Wide Coverage
  Unlike array-based methods, WGBS covers both CpG-rich and CpG-poor regions.

• Quantitative Output
  Provides methylation levels as a percentage at each site.

• Detects Non-CpG Methylation
  Especially relevant in embryonic stem cells and neurons.

• Unbiased
  Does not rely on probes or preselected regions.

Key Features of WGBS

Limitations and Challenges

• DNA Degradation
  Bisulfite treatment can fragment DNA, reducing yield and requiring optimized protocols.

• High Sequencing Cost
  Due to whole genome coverage, deep sequencing is required (~30–60x), increasing cost.

• Bioinformatics Intensive
  Requires specialized tools and high computational resources.

• Cannot Distinguish 5mC from 5hmC
  Standard WGBS detects 5-methylcytosine but not 5-hydroxymethylcytosine unless additional methods are used.

• PCR Bias
  Amplification steps can introduce bias in methylation quantification.

Popular Tools and Pipelines for WGBS Analysis

• Alignment: Bismark, BS-Seeker2, bwa-meth

• Methylation Calling: MethylDackel, MethylKit, Bismark

• Quality Control: FastQC, Qualimap, Trim Galore (bisulfite mode)

• Differential Methylation: DSS, DMRcaller, MethPipe

• Visualization: IGV (bisulfite mode), MethGo, UCSC Genome Browser

• Annotation: Bedtools, HOMER, annotatr, Bioconductor packages