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

开发一种高效的高通量技术，用于鉴定跨多种组织类型的高影响力非编码体细胞变异

• 批准号: 10662860
• 负责人: Peter J Park
• 金额: $ 44.83万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10662860
• 关键词: ATAC-seq; Address; Algorithms; Alleles; Benchmarking; Binding; Biology; Brain; Cancer Etiology; Catalogs; Cell division; Chromatin; Chromatin Structure; Computer software; Conceptions; Cost Analysis; DNA; DNA Repair; DNA purification; DNA sequencing; Data; Data Set; Detection; Development; Disease; Genetic Enhancer Element; Genome; Genomic Segment; Genomics; Goals; Human; Human Biology; Individual; Malignant Neoplasms; Methods; Modification; Mosaicism; Mutagens; Mutate; Mutation; Mutation Detection; Normal tissue morphology; Nucleic Acid Regulatory Sequences; Phase; Phenotype; Protocols documentation; Publishing; Recurrence; Research; Sampling; Sensitivity and Specificity; Site; Somatic Mutation; Techniques; Technology; Testing; Tissue Microarray; Tissues; Training; Untranslated RNA; Validation; Variant; age related; base; blastocyst; brain tissue; cell type; computerized tools; cost; cost effective; design; detection method; disorder risk; genetic variant; genome sequencing; human tissue; improved; large datasets; mosaic; promoter; scale up; targeted sequencing; technology development; tool; transcription factor; whole genome

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.

项目摘要 体细胞突变在正常组织中积累，并且越来越多地被认为是一种关键的 疾病风险的决定因素，特别是与年龄有关的疾病和癌症。体细胞突变表明 在非编码基因组的显示“开放”染色质结构的部分中富集，如活性的 启动子和增强子元件，因为开放的染色质更容易受到诱变剂的影响。此外，委员会认为， 转录因子的结合似乎阻碍了DNA修复，增加了形成固定的，双- 搁浅突变这些机制的通道效应导致体细胞的浓度增加， 基因组中有限但关键区域的突变。体细胞突变增加了 导致疾病的基因组位点经常出现反复突变，甚至在特定的基因组位点经常出现反复突变。 基础，允许开发具有更高灵敏度，更低成本和更高成本的靶向方法。 与传统的测序技术相比， 目前用于鉴定体细胞突变的方法通常利用深度（≥ 250 X）全基因组 测序（WGS），往往是昂贵的，创建大型数据集，这是计算上的挑战， 分析，并且检测具有非常低的等位基因分数的体细胞变体的能力有限。我们建议两个 阶段的方法来开发一个新的工具来解决这些缺点。在第一阶段，我们将开发一个 使用ATAC-seq检测体细胞突变的方法ATAC-seq靶向细胞的开放染色质区域， 因此，基因组集中在具有增加的体细胞突变的区域，这些区域具有增加的可能性， 具有生物学意义，仅包含一小部分基因组，从而创建更易于管理的数据集， 允许更深的测序以增加体细胞突变检测的灵敏度。这一阶段的 该提案包括三个目标：修改ATAC-seq协议，以允许检测体细胞癌。 突变;开发分析软件以分析数据;以及测试方案。 在该议定书的第二阶段，将利用第一阶段获得的数据建立一个小组 测序方案，以进一步缩小所观察的基因组区域，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）。这个阶段 还将涉及三个目标：扩大ATAC-seq分析，以确定最佳区域，包括在 测序面板;测序面板的开发;以及在一系列个体上测试面板， 组织类型。 该项目将提供快速和廉价的方法，用于检测潜在的关键体细胞 任何组织类型的突变。在研究层面，它将允许分析大量样本， 提供了生物学上重要的体细胞突变的关键信息，因此是一个重要的工具， 有助于阐明非编码基因组中的体细胞突变谱。