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到转录组的流动中，表观遗传调控在以组织特异性和暂时特异性的方式调节基因型到表型的表达方面起着关键作用。表观遗传调控是一种基本的调控机制，不仅涉及人类正常发育过程，而且涉及许多人类疾病。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甲基化是最稳定的表观遗传修饰形式，可导致转录沉默、X染色体失活和印记。在不同的正常和疾病组织中获得基因组尺度的DNA甲基化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发展相比，以合理的成本表征人类基因组中每一个CpG的甲基化状态仍然是不切实际的。本提案的目标是实现基因组尺度上非重复CpG岛DNA甲基化状态的高效和廉价的数字量化。我们计划通过开发两种互补的方法来实现这一目标，这两种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），并将这些目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），因为测序目标的大小从完整的人类基因组减少到总长度约20Mbps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一个池被捕获和测序，因此我们的方法不需要机器人设备来进行复杂的液体处理。我们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，使其广泛适应不同的DNA测序平台，从而使通量和成本在未来呈线性增长。具体目的如下：1)利用挂锁探针对人类基因组中所有非重复CpG岛进行亚硫酸氢盐单分子测序；2)基于阵列捕获CpG岛，对人类基因组中所有非重复CpG岛进行鸟枪亚硫酸盐测序；3)干细胞分化过程中整体DNA甲基化变化的表征；4)开发计算基础设施和方法，使用户能够轻松获取，评估和可视化下一代DNA测序数据，并将有助于优化亚硫酸氢盐测序技术的发展。所提出的基因组级亚硫酸氢盐测序方法将在规模（基因组中80% CpG岛）和分辨率（单个CpG，单分子）分析方面比现有的表观基因组分析技术有重大改进。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，并提供一种分类疾病亚型的可靠方法。

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.