BisulfiteSequencing

Bisulfite sequencing is a method for determining DNA methylation at single-base resolution. It relies on treatment of DNA with sodium bisulfite, which converts unmethylated cytosine residues to uracil, while methylated cytosines remain unaltered. During subsequent PCR and sequencing, uracils are amplified as thymine, allowing a readout of methylation at cytosines by comparing treated sequences with the reference genome.

Applications range from whole-genome analysis to targeted surveys. In whole-genome bisulfite sequencing (WGBS), the entire genome is sequenced after conversion. Reduced representation bisulfite sequencing (RRBS) enriches for CpG-dense regions by enzyme digestion, reducing cost. Targeted bisulfite sequencing focuses on predefined loci using locus-specific amplification.

Workflow typically involves DNA extraction, bisulfite conversion, library preparation with adapters, and sequencing. Bisulfite treatment degrades DNA and converts sequence complexity, requiring higher input amounts and careful design of primers and adapters to accommodate converted sequences. Controls are used to estimate conversion efficiency.

Data analysis uses bisulfite-aware alignment to a reference genome and methylation calling at individual CpG sites. Popular tools include Bismark and BS-Seeker. Outputs include methylation percentage per CpG and coverage. A limitation is that standard bisulfite sequencing cannot distinguish 5-methylcytosine from 5-hydroxymethylcytosine; additional methods like oxidative bisulfite sequencing (oxBS-seq) or TAB-seq can differentiate them.

Bisulfite sequencing is widely used to study epigenetic regulation, imprinting, development, and cancer. Its strengths are single-base resolution and genome-wide reach in WGBS; its limitations include DNA degradation, incomplete conversion, and analytical complexity. The technique remains a central standard in DNA methylation research.