Global comparison between methylomes of sperm cells and somatic cells

The common CGs with depth greater than 10 × among all sample were used for global comparison between methylomes of sperm cells and somatic cells. Cluster analysis, PCA analysis, and DMC detection were applied using a R package (methykit, R version 3.3.3) [89]. The DMCs were defined as the methylation difference greater than 30% and q value <0.01 between sperm cells and somatic cells. The genome structure annotation files for refGene, CGI, repeats were downloaded from the UCSC database [90]. The promoter regions were defined as ±1000 bp around the transcript start sites. The methylation levels for each element in different genomic features were calculated as the average methylation level of the CGs with at least 5 × coverage. Only the elements that met the following criteria were used for further analysis: at least 10% CG detection rate for elements with more than 50 CGs and at least five CGs detected for elements with fewer than 50 CGs. R packages were used to plot the comparison results.