The methylation level at each CpG site was calculated as the percentage of the methylated alleles over the sum of methylated and unmethylated alleles. The mean methylation level was calculated using methylation levels of all measured CpG sites within each targeted region. For quality control, each experiment included non-CpG cytosines as internal controls to verify efficient sodium bisulfite DNA conversion. We also included low, medium, high methylated standards (EpigenDx, MA) as controls in each run. In light of the low methylation values observed at some CpG sites (<4%) in the Discovery cohort, additional PCR bias testing was performed by EpigenDx for the entire ADS580 assay (i.e., all 20 CpG sites taken together), as well as individually for CpG 14. The bias testing was conducted by mixing the unmethylated DNA control and in vitro methylated DNA at different ratios (0%, 2.5%, 5%, 7.5%, 10%, 50% and 100%) followed by bisulfite modification, PCR and pyrosequencing analysis, which were run in triplicate. There was a high correlation between the percent methylation obtained from the mixing study and expected methylation percentages (r2>0.97), which confirms the quality of our data (Supplementary Fig. 1).
