Amanda J Lea, Susan C Albert, Jenny Tung, Xiang Zhou
Identifying sources of variation in DNA methylation levels is important for understanding gene regulation. Recently, bisulfite sequencing has become a popular tool for estimating DNA methylation levels at base-pair resolution, and for investigating the major drivers of epigenetic variation. However, modeling bisulfite sequencing data presents several challenges. Methylation levels are estimated from proportional read counts, yet coverage can vary dramatically across sites and samples. Further, methylation levels are influenced by genetic variation, and controlling for genetic covariance (e.g., kinship or population structure) is crucial for avoiding potential false positives. To address these challenges, we combine a binomial mixed model with an efficient sampling-based algorithm (MACAU) for approximate parameter estimation and p-value computation. This framework allows us to account for both the over-dispersed, count-based nature of bisulfite sequencing data, as well as genetic relatedness among individuals. Furthermore, by leveraging the advantages of an auxiliary variable-based sampling algorithm and recent mixed model innovations, MACAU substantially reduces computational complexity and can thus be applied to large, genome-wide data sets. Using simulations and two real data sets (whole genome bisulfite sequencing (WGBS) data from Arabidopsis thaliana and reduced representation bisulfite sequencing (RRBS) data from baboons), we show that, compared to existing approaches, our method provides better calibrated test statistics in the presence of population structure. Further, it improves power to detect differentially methylated sites: in the RRBS data set, MACAU detected 1.6-fold more age-associated CpG sites than a beta-binomial model (the next best approach). Changes in these sites are consistent with known age-related shifts in DNA methylation levels, and are enriched near genes that are differentially expressed with age in the same population. Taken together, our results indicate that MACAU is an effective tool for analyzing bisulfite sequencing data, with particular salience to analyses of structured populations. MACAU is freely available at http://www.xzlab.org/software.html.