Critical evaluation of the reliability of DNA methylation probes on the Illumina MethylationEPIC BeadChip microarrays

Abstract

DNA methylation (DNAm) plays a crucial role in a number of complex diseases. However, the reliability of DNAm levels measured using Illumina arrays varies across different probes. Previous research primarily assessed probe reliability by comparing duplicate samples between the 450k-450k or 450k-EPIC platforms, with limited investigations on Illumina EPIC arrays. We conducted a comprehensive assessment of the EPIC array probe reliability using 138 duplicated blood DNAm samples generated by the Alzheimer's Disease Neuroimaging Initiative study. We introduced a novel statistical measure, the modified intraclass correlation, to better account for the disagreement in duplicate measurements. We observed higher reliability in probes with average methylation beta values of 0.2 to 0.8, and lower reliability in type I probes or those within the promoter and CpG island regions. Importantly, we found that probe reliability has significant implications in the analyses of Epigenome-wide Association Studies (EWAS). Higher reliability is associated with more consistent effect sizes in different studies, the identification of differentially methylated regions (DMRs) and methylation quantitative trait locus (mQTLs), and significant correlations with downstream gene expression. Moreover, blood DNAm measurements obtained from probes with higher reliability are more likely to show concordance with brain DNAm measurements. Our findings, which provide crucial reliable information for probes on the EPIC array, will serve as a valuable resource for future DNAm studies.

Keywords: DNA methylation; EPIC array; probe reliability.

Figure 1

Distribution of estimated intraclass correlation coefficient (ICC) for 138 technical duplicate DNA methylation samples generated from 69 independent subjects in the ADNI study. Dashed line indicates median (red) and mean (blue) of ICC values at 0.325 and 0.381, respectively.

Figure 2

Probe reliability (ICC) increased as standard deviation (SD) of DNA methylation levels increased.

Figure 3

DNA methylation levels at cg14089881 for 69 pairs of duplicate samples in the ADNI dataset shows ICC value can be high (ICC = 0.8099) even when there is substantial disagreement in duplicate measurements (HoLA = 0.2521). In (A), each point corresponds to methylation levels of duplicate samples from the same subject, and the centerline indicates perfect agreement. In both (A) and (B), dashed lines indicate 95% confidence limits of agreement, which is computed as 1.96 x standard deviation of the differences between the two duplicate measurements. In (B), shown is histogram of the differences in methylation levels in the duplicate samples. Abbreviation: HoLA = Half-width of Limits of Agreement

Figure 4

Higher probe reliability (modified ICC) is associated with smaller absolute difference in the estimated effect sizes of DNAm-to-AD diagnosis associations in ADNI and AIBL studies (P < 2.2 × 10⁻¹⁶). The effect sizes for DNAm-to-AD associations were obtained from Silva et al. (2022) (PMID: 35982059). Reliability of the probes were determined based on modified ICC: excellent (>0.75), Good (0.6–0.75), Fair (0.4–0.6), or Poor (< 0.4).

Figure 5

CpG probes located within differentially methylated regions (DMRs) had significantly higher probe reliability (modified ICC) compared to other probes (P = 0.049). The AD-associated DMRs were obtained from Silva et al. (2022) (PMID: 35982059).