Abstract P139: Stability of DNA Methylation Profiles in Human DNA Samples in Long Term Storage

Abstract

Introductions: Historic DNA samples represent a potentially highly valuable resource for epigenetic analysis. Reduced representation bisulfite sequencing (RRBS) is a cost-effective, near genome-wide method for quantifying DNA methylation levels at single base resolution. It is unknown whether historic DNA samples stored for a long time (15-20 years) under various conditions could maintain stable methylation profiles as determined by RRBS. Methods: We used 5 groups of DNA samples (n=4 in each group) to compare and evaluate the stability of DNA methylation profiles under standard storage conditions of 4°C since 1996. Group 1 had been extracted from EDTA-anticoagulated blood and stored at 4°C since 1996; Group 2 had been diluted from group 1 to 10ng/μl and stored at -20°C since 2009; Group 3 were extracted from citrate-anticoagulated blood stored at -80°C since 1996; Group 4 were extracted from fresh EDTA-anticoagulated blood; Group 5 were extracted from fresh citrate-anticoagulated blood. The samples in groups 1, 2 and 3 were from the same subjects. All DNA samples were processed for RRBS libraries and then sequenced. Results: The global methylation level of group 1 was lower than group 2 (36.88±4.12% vs 43.66±1.29%, p=0.04), but was not statistically different from group 3 (44.31±1.46%, p=0.07). The methylation level of groups 4 and 5 (40.34±3.73% and 38.64±1.94%) was not significantly different. In addition, group 4 and 5 were not significantly different from group 1. The correlation of methylation levels for all CpG sites between individual samples in group 1 (0.987±0.001) was higher than that in group 2 and group 3 (0.984±0.002, p=0.01, and 0.969±0.015, p=0.01) and not significantly different from group 4 or 5. For CpG sites within CpG islands (CGI), the correlation between individual samples was not significantly different between any of the 5 groups. Moreover, the samples from the same subject show high correlation levels between groups 1, 2, and 3 for all CpG sites (0.987±0.006) and CpG sites within CGI (0.983±0.009). Conclusion: DNA samples stored at 4°C for prolonged periods of time are amenable to RRBS analysis. In addition, there were no significant differences in the DNA methylation profiles between citrate- and EDTA-anticoagulated blood.