Two methods were employed to determine cytosine methylation at the CpG dinucleotides. In both methods, DNA is treated with bisulfite to convert unmethylated cytosines to uracil, leaving methylated cytosines unmodified. The region containing the first CpG island was PCR amplified, and a second round of amplification was performed using nested primers. The first method employs cloning the amplified fragments, isolating individual clones, and sequencing the amplified inserts. The second method employs direct sequencing of the amplified fragment. Trace files are processed using the ESME (epigenetic sequencing methylation analysis) software (Lewin et al, 2004), which corrects trace files for quality problems, incomplete conversion, imbalanced or overscaled signals, and basecaller artifacts to provide quantitative measurement of cytosine methylation.
