High-Throughput Targeted Repeat Element Bisulfite Sequencing (HT-TREBS): Genome-Wide DNA Methylation Analysis of IAP LTR Retrotransposon

In vertebrates, DNA methylation-mediated repression of retrotransposons is essential for the maintenance of genomic integrity. In the current study, we developed a technique termed HT-TREBS (High-Throughput Targeted Repeat Element Bisulfite Sequencing). This technique is designed to measure the DNA methylation levels of individual loci of any repeat families with next-generation sequencing approaches. To test the feasibility of HT-TREBS, we analyzed the DNA methylation levels of the IAP LTR family using a set of 12 different genomic DNA isolated from the brain, liver and kidney of 4 one-week-old littermates of the mouse strain C57BL/6N. This technique has successfully generated the CpG methylation data of 5,233 loci common in all the samples, representing more than 80% of the individual loci of the five targeted subtypes of the IAP LTR family. According to the results, approximately 5% of the IAP LTR loci have less than 80% CpG methylation levels with no genomic position preference. Further analyses of the IAP LTR loci also revealed the presence of extensive DNA methylation variations between different tissues and individuals. Overall, these data demonstrate the efficiency and robustness of the new technique, HT-TREBS, and also provide new insights regarding the genome-wide DNA methylation patterns of the IAP LTR repeat elements.

在脊椎动物中，DNA甲基化介导的反转录转座子抑制作用对维持基因组完整性至关重要。本研究中，我们开发了一种命名为HT-TREBS（High-Throughput Targeted Repeat Element Bisulfite Sequencing，高通量靶向重复元件亚硫酸氢盐测序）的技术。该技术旨在借助下一代测序技术，实现任意重复序列家族单个位点的DNA甲基化水平定量检测。为验证HT-TREBS的技术可行性，我们利用12份提取自4只1周龄C57BL/6N近交系小鼠同窝幼崽的大脑、肝脏及肾脏组织的基因组DNA样本，分析了IAP LTR家族的DNA甲基化水平。该技术成功获取了所有样本共有的5233个位点的CpG甲基化数据，覆盖IAP LTR家族5个目标亚型中超过80%的单个位点。分析结果显示，约5%的IAP LTR位点的CpG甲基化水平低于80%，且无基因组位置偏好性。对IAP LTR位点的进一步分析还表明，不同组织与个体间存在广泛的DNA甲基化差异。综上，本研究数据证实了HT-TREBS新技术的高效性与稳定性，同时为解析IAP LTR重复元件的全基因组DNA甲基化模式提供了新的研究视角。