Genome-scale anaylsis of DNA methylation in CpG Islands with bisulfite sequencing

描述（由申请人提供）：在遗传信息从基因组流向转录组的过程中，表观遗传调控在以组织特异性和暂时特异性方式调节基因型至表型的表达中起关键作用。表观遗传调控是一种重要的生物学机制，它不仅参与正常的发育过程，而且参与许多人类疾病的发生。表观遗传调控可以发生在DNA的甲基化、组蛋白的共价修饰或核小体与DNA的相互作用中。DNA甲基化是表观遗传修饰的最稳定形式，其导致转录沉默、X染色体失活和印记。获得不同正常和疾病组织中DNA甲基化的基因组规模模式对于理解发育过程以及许多人类疾病的病因至关重要。然而，与人类基因组测序的快速进展相比，以合理的成本表征人类基因组中每个CpG的甲基化状态仍然是不切实际的。该提案的目标是能够有效且廉价地在基因组规模上对非重复CpG岛的DNA甲基化状态进行数字量化。我们计划通过开发两种互补的方法来实现这一目标，这些方法可以从亚硫酸氢盐转化的基因组中特异性地提取所有非重复CpG岛（或任何子集），并将这些靶标选择方法与下一代DNA测序技术相结合。这将导致测序成本的显著降低（约100倍），因为测序靶标的大小从完整的人类基因组减少到总长度约20 Mbps的所有非重复CpG岛。此外，由于所有CpG岛将通过一系列单管反应作为池被捕获和测序，因此我们的方法不需要用于复杂液体处理的机器人装置。我们的方法的简单性和可扩展性将使大量生物样品中DNA甲基化模式的真正基因组规模分析成为可能。认识到DNA测序技术的快速发展，我们还将优化目标选择前端，以广泛适应不同的DNA测序平台，从而使未来的吞吐量和成本呈线性增长。具体目标如下：1）使用挂锁探针对人类基因组中所有非重复CpG岛进行单分子亚硫酸氢盐测序; 2）基于阵列捕获CpG岛，用于对人类基因组中所有非重复CpG岛进行鸟枪亚硫酸氢盐测序; 3）表征干细胞分化中的整体DNA甲基化变化; 4）开发计算基础架构和方法，使用户能够轻松获取，评估和可视化下一代DNA测序数据，并有助于优化亚硫酸氢盐测序技术的开发。所提出的基因组规模的亚硫酸氢盐测序方法将在分析的规模（基因组中>80%的CpG岛）和分辨率（单个CpG，单个分子）方面代表对现有表观基因组分析技术的显著改进。它将加速对不同发育阶段各种组织中DNA甲基化模式的研究。它还将提供许多人类疾病中异常DNA甲基化的数字图谱，并为疾病亚型分类提供一种可靠的方法。 公共卫生相关性：表观遗传过程调节人类基因组在正常发育过程和许多病理状态（包括人类癌症）中的包装和功能。我们建议开发基因组规模的亚硫酸氢盐基因组测序方法，用于DNA甲基化的全球数字分析，以及用于分析和可视化大量亚硫酸氢盐测序数据的相关计算方法。通过靶向表观基因组捕获和下一代DNA测序的无缝集成，我们将实现整个人类基因组DNA甲基化景观的基因组规模数字分析。这项技术将有助于理解DNA甲基化在各种发育过程和人类疾病中的基因调控中的功能作用。

DESCRIPTION (provided by applicant): In the flow of genetic information from the genome to the transcriptome, epigenetic regulation plays a critical role in modulating the expression of genotypes to phenotypes in a tissue specific and temporarily specific manner. Epigenetic regulation is a fundamental mechanism that involves not only in normal developmental processes but also in many human diseases. Epigenetic regulation can take place in the methylation of DNA, covalent modifications of histone, or interactions between nucleosome and DNA. DNA methylation is the most stable form of epigenetic modification that leads to transcriptional silencing, X chromosome inactivation and imprinting. Obtaining genome-scale patterns of DNA methylation in different normal and disease tissues are critical for understanding the developmental processes as well as the etiology of many human diseases However, in contrast to rapid advances in human genome sequencing, it is still impractical to characterize the methylation status of every single CpG in the human genome at a reasonable cost. The goal of this proposal is to enable digital quantification of DNA methylation status of non-repetitive CpG islands on the genome scale efficiently and inexpensively. We plan to achieve this goal by developing two complementary methods that can specifically extract all non-repetitive CpG islands (or any subset) from the bisulfite converted genome, and coupling these target selection methods with next-generation DNA sequencing technologies. This will lead to a significant reduction (~100-fold) in the cost of sequencing, as the size of the sequencing target is reduced from the full human genome to all non-repetitive CpG islands of ~20Mbps in total length. In addition, since all CpG islands will be captured and sequenced as a pool through a series of single-tube reactions, our methods do not require robotic devices for complicated liquid handling. The simplicity and scalability of our methods will enable true genome-scale analysis of DNA methylation patterns in a large number of biological samples. Recognizing the rapid evolution of DNA sequencing technology, we will also optimize the target selection front-ends to be widely adaptable to different DNA sequencing platforms, such that the throughput and cost will scale linearly in the future. The specific aims are as follows: 1) Single molecule bisulfite sequencing of all non-repetitive CpG islands in the human genome using padlock probes; 2) array-based capture of CpG islands for shotgun bisulfite sequencing of all non-repetitive CpG islands in the human genome; 3)characterization of global DNA methylation changes in the differentiation of stem cells; 4) develop computational infractructure and methods that will enable users to easily acquire, evaluate and visualize the next-generation DNA sequencing data and will help optimize the bisulfite sequencing technology development. The proposed genome-scale bisulfite sequencing approach will represent significant improvements over existing epigenomic profiling technologies in both the scale (>80% CpG islands in the genome) and the resolution (single CpG, single molecule) of analyses. It will accelerate the studies of global DNA methylation patterns in various tissues at different developmental stages. It will also provide digital profiles of aberrant DNA methylation in many human diseases and offer a robust method for classifying disease subtypes. PUBLIC HEALTH RELEVANCE: Epigenetic processes modulate the packaging and function of the human genome in normal developmental processes and many pathologic states, including human cancers. We propose to develop genome-scale bisulfite genomic sequencing methods for global digital analysis of DNA methylation, as well as the associated computational methods for the analysis and visualization of the massive bisulfite sequencing data. Through the seamless integration of targeted epigenomic capture and next-generation DNA sequencing, we will enable genome-scale digital profiling of the DNA methylation landscape across the human genome. This enabling technology will help understand the functional roles of DNA methylation in gene regulation in various developmental processes and human diseases.