A mammalian methylation array for profiling methylation levels at conserved sequences

Abstract

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.

Universal DNA methylation age across mammalian tissues

“…With regards to marsupials, we encountered two limitations. First, less than half of the eutherian CpGs apply to marsupials 2 . Second, there were only seven marsupial species in our data set with total sample size N=162.…”

Characterizing the properties of bisulfite sequencing data: maximizing power and sensitivity to identify between-group differences in DNA methylation

“…Commercial DNAm arrays, such as the Illumina EPIC BeadChip array, are commonly utilized as an alternative strategy to bisulfite sequencing approaches in large human studies, due to their relatively low cost and the ease of interpreting data [33]. To further characterize the accuracy and sensitivity of RRBS, we performed a comparison with DNAm levels quantified using a novel Illumina Beadchip vertebrate DNAm array [30] on an overlapping set of 80 10 mouse frontal cortex DNA samples. A total of 3,552 unique DNAm sites were quantified in both the RRBS and array datasets, with each RRBS sample containing a mean of 2,263 overlapping DNAm data points (SD = 104).…”

“…A subset of 80 DNA samples were additionally profiled using a custom Illumina DNAm array (the "HorvathMammalMethylChip40" [30]). Briefly, this array includes ~36k CpGs that are located in genomic regions highly-conserved across 50 mammalian species.…”

“…Using properties derived from a large RRBS dataset generated by our group, we designed a simulation framework to explore how accuracy changes as a function of read depth, as well as comparing the DNAm level estimated from RRBS data with levels quantified using a novel Illumina array [30]. We then extended our simulation framework to investigate how statistical power to identify differences in DNAm level between groups varies as a function of read depth and sample size while also considering the effect of i) the level of DNAm at individual DNAm sites, ii) the expected difference in DNAm between groups, and iii) the balance of sample sizes between comparison groups.…”

Epigenetic clock and methylation studies in dogs

“…Further, our initial study utilized only a few canine breeds which prevented testing the relationship between epigenetic aging and breed lifespan. Here we developed a new canine epigenetic clock based on 51 recognized dog breeds, 8 and using a custom array that profiles cytosines whose flanking DNA sequences are conserved across mammalian species 9 .…”