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. 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. 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.

中文翻译：

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表观遗传年龄预测的新目标方法。

与年龄相关的DNA甲基化变化为衰老过程提供了有希望的生物标记。尽管全基因组DNA甲基化谱通过整合许多与年龄相关的CG二核苷酸（CpG）来实现可靠的年龄预测指标，但针对特定CpG进行靶向测量的方法有多种，可以更好地支持标准化且具有成本效益的高通量分析。在这项研究中，我们利用4647 Illumina BeadChip血液概况来选择CpG位点，这些位点可促进基于焦磷酸测序的可靠年龄预测。我们证明，通过液滴数字PCR（ddPCR）可以进一步提高DNA甲基化测量的精度。相比之下，亚硫酸氢盐条码扩增子测序（BBA-seq）在按年龄排序与单个CpG的DNA甲基化之间的相关性略低，而年龄预测总体上相对准确。此外，BBA-seq数据显示，邻近CpG位点的甲基化水平与年龄的相关性遵循钟形曲线，通常与CTCF结合位点相关。我们证明，在单个BBA-seq中读取的邻近CpGs处的DNA甲基化并没有被一致地修饰，而是揭示了一种随机模式。在此基础上，我们开发了一种基于个体读数中甲基化和非甲基化位点的二进制续集进行表观遗传年龄预测的新方法，该方法反映了样本中表观遗传老化的异质性。通过焦磷酸测序，BBA-seq，尤其是ddPCR，可以在几个与年龄相关的CpG上进行靶向DNA甲基化分析，从而实现表观遗传年龄预测的高精度。此外，