Evaluation of microarray-based DNA methylation measurement using technical replicates: the Atherosclerosis Risk In Communities (ARIC) Study

Maitreyee Bose
Chong Wu
James S. Pankow
Ellen W. Demerath
Jan Bressler
Myriam Fornage
Megan L. Grove
Thomas H. Mosley
Chindo Hicks
Kari North
Wen Hong Kao
Yu Zhang, College of Saint Benedict/Saint John's University
Eric Boerwinkle
Weihua Guan


Background: DNA methylation is a widely studied epigenetic phenomenon; alterations in methylation patterns influence human phenotypes and risk of disease. As part of the Atherosclerosis Risk in Communities (ARIC) study, the Illumina Infinium HumanMethylation450 (HM450) BeadChip was used to measure DNA methylation in peripheral blood obtained from similar to 3000 African American study participants. Over 480,000 cytosine-guanine (CpG) dinucleotide sites were surveyed on the HM450 BeadChip. To evaluate the impact of technical variation, 265 technical replicates from 130 participants were included in the study.

Results: For each CpG site, we calculated the intraclass correlation coefficient (ICC) to compare variation of methylation levels within- and between-replicate pairs, ranging between 0 and 1. We modeled the distribution of ICC as a mixture of censored or truncated normal and normal distributions using an EM algorithm. The CpG sites were clustered into low- and high-reliability groups, according to the calculated posterior probabilities. We also demonstrated the performance of this clustering when applied to a study of association between methylation levels and smoking status of individuals. For the CpG sites showing genome-wide significant association with smoking status, most (similar to 96%) were seen from sites in the high reliability cluster.

Conclusions: We suggest that CpG sites with low ICC may be excluded from subsequent association analyses, or extra caution needs to be taken for associations at such sites.