Use of the Illumina EPIC methylation array for epigenomic research in the crab‐eating macaque (Macaca fascicularis)

Abstract Background Commercially available Illumina DNA methylation arrays (HumanMethylation 27K, HumanMethylation450, and MethylationEPIC BeadChip) can be used for comprehensive DNA methylation analyses of not only the human genome but also other mammalian genomes, ranging from those of nonhuman primates to those of rodents. However, practical application of the EPIC array to the crab‐eating macaque has not been reported. Methods Through bioinformatic analyses involving cross‐species comparison and consideration of probe performance, we selected array probes that can be reliably used for the crab‐eating macaque genome. A DNA methylation assay using an EPIC array was performed on genomic DNA extracted from the brains of five crab‐eating macaques. The obtained DNA methylation data were compared with a publicly available dataset. Results Among the 865 918 probes in the EPIC array, a total of 183 509 probes (21.2%) were selected as high‐confidence array probes in the crab‐eating macaque. Subsequent comparisons revealed that the data from these probes showed good concordance with other DNA methylation datasets of the crab‐eating macaque. Conclusion The selected high‐confidence array probes would be useful for high‐throughput DNA methylation assays of the crab‐eating macaque.

species, ranging from nonhuman primates to rodents. 39 Typically, this was done by assessing the conservation of genomic context around the probe region between humans and the species of interest by a bioinformatic approach.
The crab-eating macaque (cynomolgus monkey, Macaca fascicularis) is a cercopithecine primate in the group of Old World monkeys and is widely distributed in Southeast Asia. Due to its ease of rearing and propagation, as well as the close relatedness of its brain structure and function to those of humans, this macaque has become one of the most widely used nonhuman primates in biomedical research.
Previously, applications of HumanMethylation27K and HumanMethylation450K arrays (Illumina), which contain approximately 27 000 and 450 000 array probes, respectively, to the crab-eating macaque have been reported. 3,8 Here, we report the application of the updated version of the array, MethylationEPIC (Illumina), containing approximately 850 000 probes, to the crab-eating macaque.

| Animals
Five cynomolgus monkeys (Macaca fascicularis; 2 males and 3 females, 34 years old) were purchased from HAMRI Co., Ltd. All procedures for animal care and experimentation were approved by the University of Tsukuba Animal Experiment Committee and were carried out in accordance with the Guidelines for Proper Conduct of Animal Experiments by the Science Council of Japan.

| Probe filtering and selection strategy
Probe selection was performed according to our previous report. 9 In brief, we used the manifest file provided by the manufacturer (Illumina). The information for SourceSeq, ProbeSeq, MapInfo, and AlleleA ProbeSeq was provided in the manifest file (Infinium MethylationEPIC v1.0 B4), which indicated the bisulfiteunconverted design sequence, the probe sequence, the coordinates of the target site, and the sequence for probe A, respectively. We retrieved human bisulfite-unconverted probe sequences (referred to as modified SourceSeq) based on the MapInfo and AlleleA ProbeSeq.
Illumina arrays include two types of array probes. Type I probe contains two probes designed for methylated and unmethylated CpG at one CpG site, whereas type II probe contains single probe for calculation of methylation level at one CpG site. For type I and type II probes, we retrieved 50 and 51 bp of the human sequence, respectively. We mapped the modified SourceSeq to the reference genome of the crab-eating macaque, Macaca_fascicularis_5.0 (mac- Scripts are available upon request.

| DNA methylation assay
Genomic DNA was extracted from frozen occipital lobes using the

| Data analysis
The IDAT file, a raw data file containing the intensities of the probes in the array, was analyzed using the minfi package 11 of R/Bioconductor and noob background correction. 12 The probes for which detection P values were <.01 across all samples (N = 5) were used. Pairwise Pearson's correlations of beta values, which are proxies for DNA methylation level and range from 0 to 1, were calculated. Average beta values were used for comparison with the published dataset. 13

| RE SULTS AND D ISCUSS I ON
Among the 865 918 probes in the HumanMethylationEPIC array, we selected the probes that could hybridize with the bisulfite-converted crabeating macaque genome (macFas5). As a result, we obtained 183 509 highconfidence probes in total ( Figure 1, Table S1). These probes account for 21.2% of all the probes in the array. In our similar calculations in the common marmoset 9 and the chimpanzee (Nakachi et al, unpublished data), we obtained approximately 9% and 44% high-confidence probes, respectively. Considering the evolutionary relationships, the number of obtained probes in this study seems reasonable. We added annotation based on macFas5 to each probe, by which one can consider orthologous relationships between humans and the crabeating macaque (Table S1). We then compared the DNA methylation values of high-confidence probes and independent DNA methylation datasets for the crab-eating macaque. Because the DNA methylation study using the occipital lobe of the crab-eating macaque has not been released, we utilized a previous study that reported methylated regions of seven different brain regions of the crab-eating macaque. 13 Within the 1310 methylated regions reported, 13 a total of 2844 high-confidence CpG probes were included. Comparison of DNA methylation levels revealed high consistency with different brain regions (R = 0.709 for the hippocampus to 0.743 for the inferior temporal lobe, average R for the seven brain regions was 0.733) (Figure 2). Similarly, both type I and type II probes also showed consistency, with average R values = .788 and .691, respectively. Because we utilized the published dataset of different brain regions of different macaques, and we did not perform validation experiments using the same brain region of same macaques, correlations remained the modest levels.
We provided high-confidence Illumina EPIC array probes that can be used in the crab-eating macaque genome. Using the well-established, low-cost platform, epigenomic research related to neuropsychiatric disorders will be facilitated in the crab-eating macaque. In particular, given the high conservation with the human genome in each array probe, the probes can be readily used for interspecies comparisons. In this study, we used the occipital lobes, however, other tissues such as blood can be applied similarly, as we demonstrated previously in other primate. 9 The limitations of DNA methylation assay using the methylation

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.

AUTH O R CO NTR I B UTI O N S
TM, MB, and K. Iwamoto designed the research. K. Ishii and TM performed the experiment. YN analyzed the data. YN, MB, TM, and K.
Iwamoto prepared the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
Raw data have been deposited in the Gene Expression Omnibus