Abstract
The reliability of methylation measures from the widely used HumanMethylation450 (HM450K) microarray has not been assessed for DNA from dried blood spots (DBS) or peripheral blood mononuclear cells (PBMC), nor for combined data from different studies. Repeated HM450K methylation measures in DNA from DBS and PBMC samples were available from participants in six case-control studies nested within the Melbourne Collaborative Cohort Study. Reliability was assessed for individual CpGs by calculating the intraclass correlation coefficient (ICC) based on technical replicates (samples repeated in a single study; 126 PBMC, 136 DBS) and study duplicates (samples repeated across studies; 280 PBMC, 769 DBS) using mixed-effects models. Reliability based on technical replicates was moderate for PBMC (median ICC = 0.42), but lower for DBS (median ICC = 0.20). Study duplicates gave lower ICCs than technical replicates. CpGs that were either highly methylated or unmethylated generally had lower ICCs, which appeared to be mostly related to their lower variability. The ICCs for global methylation measures were high, typically greater than 0.70. The reliability of methylation measures determined by the HM450K microarray is wide-ranging and depends primarily on the variability in methylation at individual CpG sites. The power of association studies is low for a substantial proportion of CpGs in the HM450K assay.
Highlights
The reliability of methylation measures from the widely used HumanMethylation[450] (HM450K) microarray has not been assessed for DNA from dried blood spots (DBS) or peripheral blood mononuclear cells (PBMC), nor for combined data from different studies
The distribution of the intraclass correlation coefficient (ICC) based on technical replicates across CpG sites is summarised in Table 1 and Fig. 1
The reliability of M-values was higher for PBMC (median ICC = 0.42, interquartile range (IQR) =0.14–0.70) compared with DBS
Summary
The reliability of methylation measures from the widely used HumanMethylation[450] (HM450K) microarray has not been assessed for DNA from dried blood spots (DBS) or peripheral blood mononuclear cells (PBMC), nor for combined data from different studies. Illumina’s product datasheet indicates “>98% reproducibility for technical replicates”, with reproducibility being defined as the correlation between replicate samples in methylation values across all CpG sites (CpG) included in the assay. This may not be an accurate reflection of the reliability of individual probes, which is a function of the variability and measurement error at each CpG. We aimed to assess the reliability of methylation measures from other sources of peripheral blood DNA (dried blood spots and mononuclear cells) and across multiple case-control studies
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