Infinium methylation arrays are not available for the vast majority of non-human mammals. Moreover, even if species-specific arrays were available, probe differences between them would confound cross-species comparisons. To address these challenges, we developed the mammalian methylation array, a single custom array that measures up to 36k CpGs per species that are well conserved across many mammalian species. We designed a set of probes that can tolerate specific cross-species mutations. We annotate the array in over 200 species and report CpG island status and chromatin states in select species. Calibration experiments demonstrate the high fidelity in humans, rats, and mice. The mammalian methylation array has several strengths: it applies to all mammalian species even those that have not yet been sequenced, it provides deep coverage of conserved cytosines facilitating the development of epigenetic biomarkers, and it increases the probability that biological insights gained in one species will translate to others.

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SUMMARYInfinium methylation arrays are widely used to robustly measure methylation of DNA in humans. However, such arrays are not available for the vast majority of non-human mammals. Moreover, even if species-specific arrays were available, probe differences between them would confound cross-species comparisons. To address these challenges, we developed the Mammalian Methylation Array, a single custom Infinium array that measures cytosine methylation levels of over 35 thousand CpG sites that are well conserved across species within the mammalian class. By design, the probes on the array tolerate cross-species mutations. To design the array, we developed the Conserved Methylation Array Probe Selector (CMAPS) algorithm, which takes as input a multi-species sequence alignment and probe design constraints. A greedy search algorithm was used to identify oligonucleotide sequences (probes) with high coverage across different mammalian species. We annotate the probes on the array with respect to genes in 159 different species and provide details on the sequence context including CpG island status and chromatin states. Our calibration experiments demonstrate the high fidelity of this array in humans, rats, and mice. The mammalian methylation array has several strengths: it applies to all mammalian species even those that have not yet been sequenced, it provides deep coverage of specific cytosines facilitating the development of highly robust epigenetic biomarkers, and it covers highly conserved CpGs which greatly increases the probability that biological insights gained in one species will readily translate to others. The mammalian methylation array is expected to find many applications in preclinical studies, comparative biology, and epigenetic studies of aging and development.

Exceptionally long-lived species, including many bats, rarely show overt signs of aging, making it difficult to determine why species differ in lifespan. Here, we use DNA methylation (DNAm) profiles from 712 known-age bats, representing 26 species, to identify epigenetic changes associated with age and longevity. We demonstrate that DNAm accurately predicts chronological age. Across species, longevity is negatively associated with the rate of DNAm change at age-associated sites. Furthermore, analysis of several bat genomes reveals that hypermethylated age- and longevity-associated sites are disproportionately located in promoter regions of key transcription factors (TF) and enriched for histone and chromatin features associated with transcriptional regulation. Predicted TF binding site motifs and enrichment analyses indicate that age-related methylation change is influenced by developmental processes, while longevity-related DNAm change is associated with innate immunity or tumorigenesis genes, suggesting that bat longevity results from augmented immune response and cancer suppression.

Aging is often perceived as a degenerative process caused by random accrual of cellular damage over time. In spite of this, age can be accurately estimated by epigenetic clocks based on DNA methylation profiles from almost any tissue of the body. Since such pan-tissue epigenetic clocks have been successfully developed for several different species, it is difficult to ignore the likelihood that a defined and shared mechanism instead, underlies the aging process. To address this, we generated 10,000 methylation arrays, each profiling up to 37,000 cytosines in highly-conserved stretches of DNA, from over 59 tissue-types derived from 128 mammalian species. From these, we identified and characterized specific cytosines, whose methylation levels change with age across mammalian species. Genes associated with these cytosines are greatly enriched in mammalian developmental processes and implicated in age-associated diseases. From the methylation profiles of these age-related cytosines, we successfully constructed three highly accurate universal mammalian clocks for eutherians, and one universal clock for marsupials. The universal clocks for eutherians are similarly accurate for estimating ages (r>0.96) of any mammalian species and tissue with a single mathematical formula. Collectively, these new observations support the notion that aging is indeed evolutionarily conserved and coupled to developmental processes across all mammalian species - a notion that was long-debated without the benefit of this new and compelling evidence.