Supporting data for "The genetics of epigenetics in the bovine pangenome era"

Most DNA methylation studies have used a single reference genome with little attention paid to the bias introduced due to the reference chosen. Genetic variation, including single nucleotide polymorphism (SNPs) and structural variants (SVs), can lead to differences in methylation sites (CpGs) between individuals of the same species. We analysed whole genome bisulfite sequencing (WGBS) data from the fetal liver of Angus (<i>Bos taurus taurus</i>), Brahman (<i>Bos taurus indicus</i>) and reciprocally crossed samples. Using reference genomes for each breed from the Bovine Pangenome Consortium, we investigated the influence of reference genome choice on the breed- and parent-of-origin effects in methylome analyses. <br>Our findings revealed that about 75% of CpG sites were shared between Angus and Brahman, ~5% were breed-specific, and ~20% were unresolved. We demonstrated up to 2% quantification bias in global methylation when an incorrect reference genome was used. Furthermore, we found that SNPs and SVs were 8-fold (p-value &lt; 5×10^(-324)) and 1.13-fold (p-value &lt; 5×10^(-324)) higher in CpGs, respectively, compared to the rest of the genome. We found a poor association between differentially methylated regions (DMRs) and differentially expressed genes (DEGs) and suggest that DMRs may be impacting enhancers that target these DEGs. DMRs overlapped with imprinted genes, of which one, <i>Dgat1</i>, which is important for fat metabolism and weight gain, was found in the breed-specific and sire-of-origin comparisons. <br>This work demonstrates the need to consider reference genome effects to explore genetic and epigenetic differences accurately and identify DMRs involved in controlling certain genes.

绝大多数DNA甲基化（DNA methylation）研究均采用单一参考基因组，却极少关注因所选参考基因组引入的偏差。遗传变异包括单核苷酸多态性（single nucleotide polymorphism, SNPs）与结构变异（structural variants, SVs），可导致同一物种不同个体间甲基化位点（CpGs）的差异。我们分析了安格斯牛（*Bos taurus taurus*）、婆罗门牛（*Bos taurus indicus*）及其正反交样本的胎儿肝脏组织的全基因组亚硫酸氢盐测序（whole genome bisulfite sequencing, WGBS）数据。借助牛泛基因组联盟（Bovine Pangenome Consortium）为各品种构建的参考基因组，我们探究了参考基因组选择对甲基化组（methylome）分析中品种及亲本起源效应的影响。
研究结果显示，安格斯牛与婆罗门牛间约75%的CpG位点共通，约5%为品种特异性位点，另有约20%无法确定归属。我们证实，若使用错误的参考基因组，全局甲基化水平的定量偏差可达2%。此外，我们发现CpG区域内的SNPs与SVs丰度分别较基因组其余区域高出8倍（P值<5×10^-324）与1.13倍（P值<5×10^-324）。我们发现差异甲基化区域（differentially methylated regions, DMRs）与差异表达基因（differentially expressed genes, DEGs）之间的关联度较低，并推测DMRs可能通过调控靶向这些DEGs的增强子发挥作用。DMRs与印记基因（imprinted genes）存在重叠，其中在品种特异性及父本起源比较分析中发现的*Dgat1*基因，对脂肪代谢与体重增长具有重要作用。本研究表明，若要精准探究遗传与表观遗传差异并鉴定调控特定基因的DMRs，必须考量参考基因组带来的影响。