Deep qualitative methylation analysis

Genome wide methylation analysis is limited by low coverage and the inability to detect single variants below 10%. Quantitative analysis provides accurate information on the extent of methylation of single CpG, but it does not measure the actual polymorphism of methylation profiles of single molecules. We deep sequenced bisulfite-treated DNA and analyzed the methylation profiles of a GFP gene before or after formation of a double strand break and repair by homologous recombination or non-homologous end-joining The primary DNA sequence of all molecules analyzed was identical. In contrast, the methylation profiles, not the extent of methylation, were profoundly different when we compared untreated DNA molecules with molecules subjected to DSB and repaired by HR or NHEJ. We conclude that DNA damage and repair are the primary source of methylation polymorphism. These leave stable methylation signatures that characterize polymorphic epialleles.