Agreement in DNA methylation levels from the Illumina 450K array across batches, tissues, and time

Abstract

Epigenome-wide association studies (EWAS) have focused primarily on DNA methylation as a chemically stable and functional epigenetic modification. However, the stability and accuracy of the measurement of methylation in different tissues and extraction types is still being actively studied, and the longitudinal stability of DNA methylation in commonly studied peripheral tissues is of great interest. Here, we used data from two studies, three tissue types, and multiple time points to assess the stability of DNA methylation measured with the Illumina Infinium HumanMethylation450 BeadChip array. Redundancy analysis enabled visual assessment of agreement of replicate samples overall and showed good agreement after removing effects of tissue type, age, and sex. At the probe level, analysis of variance contrasts separating technical and biological replicates clearly showed better agreement between technical replicates versus longitudinal samples, and suggested increased stability for buccal cells versus blood or blood spots. Intraclass correlations (ICCs) demonstrated that inter-individual variability is of similar magnitude to within-sample variability at many probes; however, as inter-individual variability increased, so did ICC. Furthermore, we were able to demonstrate decreasing agreement in methylation levels with time, despite a maximal sampling interval of only 576 days. Finally, at 6 popular candidate genes, there was a large range of stability across probes. Our findings highlight important sources of technical and biological variation in DNA methylation across different tissues over time. These data will help to inform longitudinal sampling strategies of future EWAS.

Keywords: Intraclass correlations; methylation; redundancy analysis; replication; tissue stability.

Figures

Figure 1.

RDA results in the Dutch data. Based on a random sample of 100,000 probes. The symbols represent the different tissue types and the colors differentiate the 10 individuals. a. Biological replicates: repeated samplings from the same individuals. b. Technical replicates. c. Technical replicates, where RDA analysis has been adjusted for tissue type and sex. The only male in the sample is patient 8 (P8) represented in red.

Figure 2.

RDA analysis of Canadian study. Based on a random sample of 100,000 probes. The symbols represent the sex and colors represent the age. a. Biological replicates. b. Technical replicates. c. and d. Technical replicates, where RDA analysis has been adjusted for age and sex. In c, colored by age and in d, colored by individual. (Two samples originating from the same tissue samples of an individual will be of the same color in d).

Figure 3.

Distributions of intraclass correlations in the Dutch data. For different tissue types, combinations of tissue types, and for biological versus technical replicates, based on a random sample of 100,000 probes selected from 449,059 probes where the maximum methylation level was lower than 0.9 and the minimum methylation level was higher than 0.1.

Figure 4.

Summary smoothed histograms of F-statistics by replicate type. An F statistic was constructed for each methylation probe and each type of replicate using carefully constructed contrasts (see Methods section for more details). The null hypothesis for each F statistic is that the within-sample, between replicate differences in methylation demonstrate no excess variability. a. Dutch study. b. Canadian study.

Figure 5.

Venn diagram of overlap between probes demonstrating instability. Number of probes showing evidence of poor replicability via significant P values (P<0.05) a. across the three tissues for the technical replicates of the Dutch study. b. within or between batches of the technical replicates from the Canadian study.

Figure 6.

Association between sums of squares and elapsed time. For each individual in the Dutch study (a) or the Canadian study (b), the 75th percentile, across all probes, of the sums of squares measuring agreement is shown as a function of the time elapsed between the biological replicates. Note that we only used whole-blood samples in the Dutch study, since it was the only tissue for which there was a range of elapsed times between repeated samplings.

Figure 7.

RDA analysis of elapsed time among biological replicates based on random sample of 100,000 probes. a. Dutch study; b. Canadian study. We are looking at the difference in DNA methylation between two samples from an individual (taken at two different time points), therefore there is only one point per individual. For panel b, we have colored the individuals based on their age at the second time point. The shape of the symbol in panel b represents the batch corresponding to first time point (since all samples for the second time point were collected in the third batch of analyzed samples).

Figure 8.

Replicate agreement at 6 candidate genes in the Dutch study. Agreement is shown by -log10 P values derived from F-statistic measures of variability at 6 candidate genes. Each color represents a different probe in the candidate gene. The number of probes present in a gene is indicated in parenthesis beside the name of the gene. Different types of replicates or tissues are indicated along the X-axis.