Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution

Reduced-representation bisulfite sequencing, optimized for DNA amounts as low as 30 nanograms and robust enough to process DNA extracted from formalin-fixed, paraffin-embedded tissue, allows genome-scale mapping of DNA methylation in many samples. Bisulfite sequencing measures absolute levels of DNA methylation at single-nucleotide resolution, providing a robust platform for molecular diagnostics. We optimized bisulfite sequencing for genome-scale analysis of clinical samples: here we outline how restriction digestion targets bisulfite sequencing to hotspots of epigenetic regulation and describe a statistical method for assessing significance of altered DNA methylation patterns. Thirty nanograms of DNA was sufficient for genome-scale analysis and our protocol worked well on formalin-fixed, paraffin-embedded samples.