Development of an Efficient High Throughput Technique for the Identification of High-Impact Non-Coding Somatic Variants Across Multiple Tissue Types

项目总结 体细胞突变在正常组织中积累，并日益被认为是至关重要的 疾病风险的决定因素，特别是在与年龄相关的疾病和癌症中。体细胞突变显示 富含在基因组的非编码部分，显示出“开放”的染色质结构，如活性 启动子和增强子元件，因为开放染色质更容易受到诱变剂的影响。此外， 转录因子结合似乎阻碍DNA修复，增加形成固定的，双倍的 搁浅的突变。这些机制的通道效应导致了体细胞的集中 基因组中有限但关键区域的突变。体细胞突变的可能性增加 致病经常发生在重复的基因组位置，甚至经常是在特定的重复突变。 基础，允许以更高的灵敏度、更低的成本和更高的灵敏度开发目标方法 与传统的测序技术相比，提高了识别体细胞突变的能力。 目前鉴定体细胞突变的方法一般利用深部(≥250X)全基因组 测序(WGS)，而且往往很昂贵，创建的大型数据集在计算上具有挑战性 分析，并且检测等位基因比例很低的体细胞变异的能力有限。我们建议两个 采取分阶段办法，开发一种新的工具来解决这些缺点。在第一阶段，我们将开发一种 利用ATAC-seq检测体细胞突变的方法。Atac-seq靶向于细胞的开放染色质区域 基因组SO集中在体细胞突变增加的区域，这些区域有更高的可能性 在生物学上有意义，只包含基因组的一小部分，创建了更易于管理的数据集，并且 允许更深的测序，以提高体细胞突变检测的灵敏度。这一阶段 提案包括三个目标：修改atac-seq议定书，以允许检测体细胞 突变；开发分析软件来分析数据；以及测试协议。 在议定书的第二阶段，从第一阶段获得的数据将用于开发小组 测序协议，以进一步缩小所查看的基因组区域，降低分析成本，并允许 用于直接分析提取的DNA(而不是ATAC-SEQ所需的完整染色质)。这一阶段 还将涉及三个目标：扩大ATAC-SEQ分析，以确定要纳入 测序仪表板；开发测序仪表板；对一系列个人和 组织类型。 该项目将为潜在的关键体细胞的检测提供快速和廉价的方法 任何组织类型的突变。在研究层面上，它将允许对大量样本进行分析，以 提供关于生物上重要的体细胞突变的关键信息，从而成为一个重要的工具 有助于阐明非编码基因组中的体细胞突变谱。

Project summary Somatic mutations accumulate in normal tissues and are increasingly recognized as a crucial determinant of disease risk, especially in age-related conditions and cancer. Somatic mutations show enrichment in portions of the noncoding genome that show “open” chromatin structure, such as active promoter and enhancer elements, because open chromatin is more vulnerable to mutagens. Furthermore, transcription factors binding appears to obstruct DNA repair, increasing the likelihood of forming fixed, double- stranded mutations. The channeling effects of these mechanisms result in a concentration of somatic mutations in restricted, yet critical, regions of the genome. Somatic mutations with an increased likelihood of causing diseases frequently arise at recurrent genomic sites, and often even recurrent mutations at specific bases, allowing for the development of targeted methods with greater sensitivity, lower cost, and higher throughput to identify somatic mutations than traditional sequencing techniques. Present methods for identifying somatic mutations generally utilize deep (≥250X) whole genome sequencing (WGS) and tend to be expensive, create large datasets that are computationally challenging to analyze, and have limited ability to detect somatic variants with very low allele fractions. We propose a two phase approach to developing a new tool to address these shortcomings. In the first phase we will develop a method of detecting somatic mutations using ATAC-seq. ATAC-seq targets the open chromatin regions of the genome so is focused on regions with increased somatic mutations that have an increased likelihood of being biologically meaningful, only incorporates a fraction of the genome creating a more manageable dataset, and allows for deeper sequencing to increase the sensitivity of somatic mutation detection. This phase of the proposal includes three aims: modification of the ATAC-seq protocol to allow for detection of somatic mutations; development of analysis software to analyze the data; and testing of the protocol. In the second phase of the protocol, data obtained from phase one will be used to develop a panel sequencing protocol to further narrow the genomic regions looked at, reduce the cost of the analysis, and allow for extracted DNA to be directly analyzed (rather than the intact chromatin needed for ATAC-seq). This phase will also involve three aims: expansion of ATAC-seq analysis to determine the best regions to include on the sequencing panel; development of the sequencing panel; and testing of the panel on a range of individuals and tissue types. This project will provide rapid and inexpensive methods for the detection of potentially critical somatic mutations in any tissue type. At a research level, it will allow for the analysis of a large number of samples to provide critical information on biologically important somatic mutations and thus be an important tool that will help illuminate the spectrum of somatic mutation in the noncoding genome.