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.

我们在开发研究体细胞突变的实验和计算工具方面拥有十年的经验 揭示了跨多个组织的体细胞突变的时间、细胞类型特异性和机制模式 并贯穿人类的一生。然而，由于成本原因，我们对体细胞突变的看法受到限制。领域 体细胞突变需要一种具有等位基因和分子特异性的大块组织灵活性的技术 单细胞技术的力量。 为了建立人类基因组的统一体细胞变异目录，拟议的研究旨在开发 基于 Tn5 的双工测序 (Tn5-duplex-seq) 方法，一种可扩展的单孔工作流程，用于捕获 使用 Tn5 从一系列细胞或 DNA 输入中获得单分子水平的体细胞突变景观 转座酶，可通过基于简单 PCR 的捕获的链方向解析互补链。 双链测序，捕获单分子中的互补链信息，提供了前所未有的 体细胞突变检测的准确性，但可能容易出错或费力，具体取决于检测方法 捕获互补链。 Tn5-duplex-seq的核心概念已成功应用于单 细胞具有高精度（META-CS），但需要优化。在拟议的研究中，我们的目标是 1）确定 混合细胞和批量 DNA 测序的参数，以保留单分子信息和成本 有效性，以及 2) 利用单分子双链检测的力量，为套件提供基础 评估不同类型体细胞突变的分析工具，包括单核苷酸变异、插入缺失、 高精度的微卫星和拷贝数变异。 Tn5-duplex-seq 可以通过先天性改变我们对体细胞突变的理解 该技术的全面性：捕获克隆以及最罕见的体细胞突变并提供 确定体细胞单碱基和结构变异的分析方法的准确性的基础。 最终，我们的工具将易于使用、具有成本效益且可扩展，可供更大的科学界使用 并可轻松与跨人体组织的体细胞镶嵌 (SMaHT) 网络的管道集成。肌钙蛋白5- 全面分析基因突变的双工序列有可能回答基本问题 关于人类生物学和医学中的基因组。 这项研究将揭示体细胞突变的情况及其在人体组织中的积累 单分子分辨率，能够使用以下方法检测克隆和细胞特异性体细胞突变 灵活的输入工作流程。拟议的研究对于全面、基于结果的研究具有重要意义 制定发现人类发展中的突变负担和景观的策略 老化。与突变特征的计划表征一起，预期结果应该 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。