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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

基本信息

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
项目类别：
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

项目摘要

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.

我们在研究体细胞突变的实验和计算工具方面的十年经验