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

Epigenetics. 2018;13(1):19-32. doi: 10.1080/15592294.2017.1411443. Epub 2018 Jan 30.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Brain-Derived Neurotrophic Factor / genetics
  • Canada
  • Catechol O-Methyltransferase / genetics
  • DNA Methylation*
  • Dried Blood Spot Testing / methods
  • Dried Blood Spot Testing / standards
  • Epigenesis, Genetic
  • Female
  • Humans
  • Male
  • Mouth Mucosa
  • Netherlands
  • Oligonucleotide Array Sequence Analysis / methods*
  • Oligonucleotide Array Sequence Analysis / standards*
  • Oligonucleotide Array Sequence Analysis / statistics & numerical data
  • Receptors, Dopamine D2 / genetics
  • Receptors, Dopamine D4 / genetics
  • Receptors, Oxytocin / genetics
  • Reproducibility of Results
  • Serotonin Plasma Membrane Transport Proteins / genetics
  • Time Factors


  • Brain-Derived Neurotrophic Factor
  • DRD2 protein, human
  • DRD4 protein, human
  • OXTR protein, human
  • Receptors, Dopamine D2
  • Receptors, Oxytocin
  • SLC6A4 protein, human
  • Serotonin Plasma Membrane Transport Proteins
  • Receptors, Dopamine D4
  • BDNF protein, human
  • COMT protein, human
  • Catechol O-Methyltransferase