Mammalian methylation array data analysis: A practical workflow for preprocessing, quality control, and EWAS

초록

Background: The HorvathMammalMethylChip40 enables standardized DNA methylation profiling across diverse mammalian species by targeting evolutionarily conserved CpG sites; appropriate preprocessing and quality control are essential for reliable downstream analysis. Methods: This technical protocol describes a reproducible Mammalian Methylation Array workflow using SeSAMe for preprocessing and limma for epigenome-wide association study (EWAS); the workflow was demonstrated using blood-derived methylation data from 826 domestic dogs and 279 cattle to assess age-, sex-, and population-associated methylation patterns. Results: The processed Korean dog samples exhibited generally consistent bimodal β-value distributions. EWAS identified 16,275 age-associated and 1,702 sex-associated CpG sites in dogs, whereas 20,508 CpG sites were significantly associated with age in cattle. In addition, 230 CpG sites exhibited significant age-by-group interaction effects between Korean and U.S. dogs, indicating population differences in age-associated methylation patterns. Conclusion: This protocol provides a practical framework for preprocessing Mammalian Methylation Array data and identifying phenotype-associated CpG sites, thereby supporting downstream genomic annotation and functional interpretation.

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