bisulietsequencing

Bisulfite sequencing, sometimes spelled bisulite sequencing, is a method for determining DNA methylation status at single-base resolution. It relies on treating genomic DNA with sodium bisulfite, which converts unmethylated cytosines to uracil (read as thymine after PCR), while methylated cytosines remain unaltered. This selective conversion creates sequence differences that reflect the methylation pattern when the treated DNA is sequenced and compared to an unconverted reference.

A typical workflow comprises DNA extraction, bisulfite treatment, amplification and library preparation, sequencing, and bioinformatic analysis. Because unmethylated cytosines are read as thymine, the resulting reads must be aligned to a reference genome that has been transformed to accommodate C to T changes. Methylation at cytosine positions is inferred from the proportion of times a C is observed at that site in sequencing data. Variants of the method include whole-genome bisulfite sequencing (WGBS), reduced representation bisulfite sequencing (RRBS), and targeted or amplicon-based bisulfite sequencing.

Applications are broad in epigenetics and cancer research, enabling researchers to map cytosine methylation across genomes or in specific regions, and to compare methylation patterns across tissues, developmental stages, or disease states. Strengths include single-base resolution and genome-wide or targeted coverage depending on the approach. Limitations arise from DNA degradation during bisulfite treatment, incomplete conversion, potential false positives or negatives, and the need for specialized alignment and methylation-calling algorithms. Data interpretation must account for non-CpG methylation contexts in certain species and the possibility of technical biases.