New targeted approaches for epigenetic age predictions

BMC Biol. 2020 Jun 24;18(1):71. doi: 10.1186/s12915-020-00807-2.


Background: Age-associated DNA methylation changes provide a promising biomarker for the aging process. While genome-wide DNA methylation profiles enable robust age-predictors by integration of many age-associated CG dinucleotides (CpGs), there are various alternative approaches for targeted measurements at specific CpGs that better support standardized and cost-effective high-throughput analysis.

Results: In this study, we utilized 4647 Illumina BeadChip profiles of blood to select CpG sites that facilitate reliable age-predictions based on pyrosequencing. We demonstrate that the precision of DNA methylation measurements can be further increased with droplet digital PCR (ddPCR). In comparison, bisulfite barcoded amplicon sequencing (BBA-seq) gave slightly lower correlation between chronological age and DNA methylation at individual CpGs, while the age-predictions were overall relatively accurate. Furthermore, BBA-seq data revealed that the correlation of methylation levels with age at neighboring CpG sites follows a bell-shaped curve, often associated with a CTCF binding site. We demonstrate that within individual BBA-seq reads the DNA methylation at neighboring CpGs is not coherently modified, but reveals a stochastic pattern. Based on this, we have developed a new approach for epigenetic age predictions based on the binary sequel of methylated and non-methylated sites in individual reads, which reflects heterogeneity in epigenetic aging within a sample.

Conclusion: Targeted DNA methylation analysis at few age-associated CpGs by pyrosequencing, BBA-seq, and particularly ddPCR enables high precision of epigenetic age-predictions. Furthermore, we demonstrate that the stochastic evolution of age-associated DNA methylation patterns in BBA-seq data enables epigenetic clocks for individual DNA strands.

Keywords: Aging; Amplicon sequencing; Blood; Buccal swabs; CTCF; DNA methylation; Droplet digital PCR; Epigenetic; Human.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aging / genetics*
  • Blood / metabolism
  • DNA Methylation*
  • Epigenesis, Genetic / physiology*
  • Epigenomics / methods*
  • Genetic Markers
  • High-Throughput Nucleotide Sequencing*
  • Humans


  • Genetic Markers