Detection and Characterization of Somatic Mutations in Human Tissue Utilizing Duplex-Consensus Sequencing

利用双重一致性测序检测和表征人体组织中的体细胞突变

• 批准号: 10662935
• 负责人: Sangita Choudhury
• 金额: $ 44.25万
• 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/10662935
• 关键词: Aging; Alleles; Benchmarking; Bioinformatics; Brain; Cataloging; Catalogs; Cell Nucleus; Cells; Communities; Consensus; Copy Number Polymorphism; DNA; DNA Repair; DNA Sequence Alteration; DNA sequencing; Data; Data Analyses; Detection; Development; Disease Progression; Genome; Germ Layers; Heart; Human; Human Biology; Human Development; Human Genome; Individual; Informatics; Institution; Knowledge; Longevity; Medicine; Methods; Microsatellite Repeats; Mosaicism; Mutation; Mutation Spectra; Nature; Nucleotides; Pattern; Phase; Preparation; Procedures; Quality Control; Research; Resolution; Sampling; Single Nucleotide Polymorphism; Somatic Mutation; Standardization; Techniques; Technology; Technology Transfer; Tissues; Tn5 transposase; Validation; Variant; analytical method; analytical tool; base; cell preparation; cell type; computerized tools; cost; cost effective; cost effectiveness; data analysis pipeline; experience; flexibility; genetic profiling; human tissue; insertion/deletion mutation; preservation; prevent; rare variant; scale up; single molecule; technology development; tool

Our decade of experience in developing experimental and computational tools to study somatic mutations has revealed temporal, cell-type specific, and mechanistic patterns of somatic mutations across multiple tissues and across the human lifespan. Yet our view of somatic mutations has been limited due to cost. The field of somatic mutations requires a technique with the flexibility of bulk tissue with the allele- and molecule-specific power of single-cell techniques. To build the unified somatic variants catalog of human genome the proposed research aims to develop a Tn5 based duplex sequencing (Tn5-duplex-seq) method, a scalable single-well workflow to capture the landscape of somatic mutations at the single-molecule level from a range of cell or DNA input using Tn5 transposase, which resolves complementary strands by strand orientation from simple PCR-based capture. Duplex sequencing, the capture of complementary strand information in single molecules, offers unprecedented accuracy for detection of somatic mutations, but can be error-prone or laborious depending on the method of capturing complementary strands. The core concept of Tn5-duplex-seq has been successfully applied to single- cells with high accuracy (META-CS) but requires optimization. In the proposed study we aim to 1) determine parameters for pooled cell and bulk DNA sequencing to retain single-molecule information and cost- effectiveness, and 2) harness the power of single-molecule duplex detection which provides the basis for a suite of analytical tools to evaluate different types of somatic mutation including single nucleotide variants, indels, microsatellites and copy number variants with high accuracy. Tn5-duplex-seq can transform our understanding of somatic mutations through the innate comprehensiveness of this technique: capturing both clonal as well as the rarest somatic mutations and providing the basis of accuracy for analytical approaches to determine both somatic single base and structural variation. Ultimately, our tool will be accessible, cost-effective, and scalable to be used by the larger scientific community and easily integrated with pipelines of the Somatic Mosaicism across Human Tissues (SMaHT) Network. Tn5- duplex-seq with comprehensive profiling of genetic mutations has the potential to answer fundamental questions about our genomes in human biology and medicine. This study will reveal the landscape of somatic mutations and their accumulation in human tissue at single-molecule resolution, enabling detection of both clonal and cell-specific somatic mutations using a flexible-input workflow. The proposed research is significant for the comprehensive, results-based development of strategies for discovering the mutational burden and landscape in human development and aging. Together with the planned characterization of mutational signatures, the anticipated results should provide knowledge that may help to develop new strategies for preventing aging and disease progression.

我们在开发研究体突变的实验和计算工具方面的十年经验 已经揭示了多个组织中体细胞突变的时间，细胞类型特异性和机械模式 以及整个人类的寿命。然而，由于成本，我们对躯体突变的看法受到限制。领域 体细胞突变需要具有具有等位基因和分子特异性的散装组织灵活性的技术 单细胞技术的力量。 为了建立人类基因组的统一体型变体目录拟议的研究旨在发展 基于TN5的双工测序（TN5-Duplex-Seq）方法，可伸缩的单孔工作流程以捕获 使用TN5从一系列单元或DNA输入的单分子水平的体细胞突变的景观 转座酶，通过基于简单的PCR捕获从链方向解决互补的链。 双工测序，单分子中互补链信息的捕获，提供了前所未有的 检测体突变的准确性，但可能是错误的或费力的，具体取决于 捕获互补的链。 TN5二链序列的核心概念已成功地应用于单一 高精度（元CS）的细胞需要优化。在拟议的研究中，我们的目标是1）确定 合并细胞和大量DNA测序的参数，以保留单分子信息和成本 有效性，以及2）利用单分子双工检测的力量，这为套件提供了基础 分析工具评估不同类型的体细胞突变，包括单核苷酸变体，Indels， 具有高精度的微卫星和复制号变体。 tn5二链式seq可以通过先天而改变我们对躯体突变的理解 这项技术的全面性：捕获克隆和最稀有的躯体突变并提供 分析方法的准确性基础，以确定躯体单碱基和结构变化。 最终，我们的工具将被访问，具有成本效益和可扩展性，可供大型科学界使用 并很容易与人体组织（SMAHT）网络的体细胞镶嵌的管道集成。 TN5- 对基因突变进行全面分析的复合式序列有可能回答基本问题 关于我们在人类生物学和医学领域的基因组。 这项研究将揭示体细胞突变的景观及其在人体组织中的积累 单分子分辨率，可以使用克隆和细胞特异性的体细胞突变检测 灵活的输入工作流程。拟议的研究对于综合，基于结果的研究很重要 制定发现人类发展中突变负担和景观的策略和 老化。以及计划的突变特征的表征，预期的结果应 提供可能有助于制定预防衰老和疾病进展的新策略的知识。