Targeted methods for epigenetic age predictions in mice

Age-associated DNA methylation reflects aspect of biological aging—therefore epigenetic clocks for mice can elucidate how the aging process in this model organism is affected by specific treatments or genetic background. Initially, age-predictors for mice were trained for genome-wide DNA methylation profiles and we have recently described a targeted assay based on pyrosequencing of DNA methylation at only three age-associated genomic regions. Here, we established alternative approaches using droplet digital PCR (ddPCR) and barcoded bisulfite amplicon sequencing (BBA-seq). At individual CG dinucleotides (CpGs) the correlation of DNA methylation with chronological age was slightly higher for pyrosequencing and ddPCR as compared to BBA-seq. On the other hand, BBA-seq revealed that neighboring CpGs tend to be stochastically modified at murine age-associated regions. Furthermore, the binary sequel of methylated and non-methylated CpGs in individual reads can be used for single-read predictions, which may reflect heterogeneity in epigenetic aging. In comparison to C57BL/6 mice the single-read age-predictions using BBA-seq were also accelerated in the shorter-lived DBA/2 mice, and in C57BL/6 mice with a lifespan quantitative trait locus of DBA/2 mice. Taken together, we describe alternative targeted methods for epigenetic age predictions that provide new perspectives for aging-intervention studies in mice.