Ultra-High Fidelity Single-Molecule Profiling of Mosaic Double- and Single-Strand DNA Mutations and Damage

镶嵌双链和单链 DNA 突变和损伤的超高保真度单分子分析

• 批准号: 10657882
• 负责人: Gilad David Evrony
• 金额: $ 43.8万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-10 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10657882
• 关键词: Achievement; Address; Affect; Aging; Automation; Autopsy; Biological Assay; Body part; Catalogs; Categories; Cells; Characteristics; Cloud Computing; Collection; Cost Effectiveness Analysis; DNA; DNA Damage; DNA Modification Process; DNA Repair Disorder; DNA Sequence; DNA Sequence Alteration; DNA amplification; DNA sequencing; Data; Detection; Development; Dissection; Elements; Engineering; Ensure; Environmental Health; Event; Genetic Anticipation; Genetic Diseases; Genome; Goals; Human body; In Vitro; Kinetics; Laboratories; Libraries; Liquid substance; Malignant Neoplasms; Measures; Methods; Microdissection; Modification; Morphologic artifacts; Mosaicism; Mutagens; Mutate; Mutation; Oligonucleotides; Participant; Pattern; Phase; Physiology; Polymerase; Preparation; Process; Reproducibility; Research; Retroelements; Robot; Role; Sampling; Single-Stranded DNA; Source; Standardization; Technology; Testing; Time; Tissue Sample; Tissues; Validation; Variant; Work; base; comparative efficacy; computational pipelines; cost; deep neural network; design; ds-DNA; human tissue; improved; innovation; insertion/deletion mutation; machine learning model; mosaic; mosaic variant; new technology; parallelization; portability; repaired; single cell sequencing; single molecule; stem; tissue repair

PROJECT SUMMARY/ABSTRACT Somatic mosaic mutations accumulate over time in every healthy cell but detecting them requires specialized sequencing technologies with extremely low error rates. However, all current technologies for profiling mosaic mutations require amplification of DNA, which introduces single-strand DNA artifacts. Therefore, even the highest fidelity technologies can only detect mosaic mutations when they are present in both strands of the original DNA, but they cannot detect the single-strand mutations and damage from which they originate. Here, we develop a technology that can directly sequence DNA molecules without any amplification at ultra-high fidelity, such that mutations and damage present in only one of the two strands of a DNA molecule can be detected for the first time. It achieves this by significantly increasing the accuracy of single-molecule DNA sequencing, and furthermore, it utilizes long reads that can be used to study regions of the genome that are not accessible to all prior high-fidelity mosaic mutation technologies that utilize short reads. Our technology, called Hairpin Duplex Enhanced Fidelity Sequencing (HiDEF-seq), will be developed as part of the SMaHT Network, and we will work in close coordination with the Network at all stages of the project to ensure it contributes significantly to the Network’s goals of creating a comprehensive catalogue of somatic mosaicism in human tissues. In the first UG3 phase of the project, we will develop our technology to cost-effectively and reliably profile any bulk human tissue. In Aim 1 of UG3, we will develop the technology to profile all classes of single- and double-strand mosaic mutations at ultra-high fidelity (substitutions, insertions, deletions, structural variants, and retroelements). In Aim 2 of UG3, we will use machine-learning models of single-molecule polymerase kinetics to detect diverse types of single-strand DNA damage and modifications. Importantly, HiDEF-seq will achieve detection of all these events simultaneously in one assay. In the second UH3 phase of the project, we will work closely and integrally with the SMaHT Network to validate and scale the throughput of the technology so that it can profile the entire collection of SMaHT tissue samples. In Aim 1 of UH3, we will fully automate the laboratory component of HiDEF-seq to enable creation of sequencing libraries for hundreds of samples per day. In Aim 2 of UH3, we will scale the computational pipeline of our technology for rapid analysis of thousands of samples. Throughout this project, we will work with the SMaHT Network to validate, standardize, and disseminate the technology. HiDEF-seq’s achievement of ultra-high fidelity sequencing of single-strand DNA mutations and damage will enable fundamentally new types of mosaic mutation studies that will disentangle the interrelated processes of DNA mutation, repair and replication. It will also enable systematic dissection of sources of artifacts stemming from laboratory processing of DNA. Furthermore, it will reveal the instantaneous effects and temporal dynamics of exogenous mutagens, with broad implications for environmental health and discovery of factors that reduce or increase the rate at which our genomes mutate.

项目摘要/摘要 体细胞嵌合突变随着时间的推移在每个健康细胞中积累，但检测它们需要专门的 错误率极低的测序技术。然而，目前所有用于分析马赛克的技术 突变需要DNA的扩增，这会引入单链DNA伪像。因此，即使 最高保真的技术只能检测到镶嵌突变时，他们都存在于两个链的 它们可以检测原始DNA，但不能检测出它们所起源的单链突变和损伤。在这里， 我们开发了一种技术，可以直接测序DNA分子，而无需在超高温度下进行任何扩增， 保真度，使得DNA分子的两条链中仅存在一条链中的突变和损伤， 第一次被发现。它通过显著提高单分子DNA的准确性来实现这一点 此外，它利用长读段，可用于研究基因组的区域， 不能被所有现有的利用短读段的高保真镶嵌突变技术所利用。我们的技术， 称为发夹双链增强保真测序（HiDEF-seq），将作为SMaHT的一部分开发 我们将在项目的各个阶段与该网络密切协调，以确保 大大有助于网络的目标，创造一个全面的目录体细胞镶嵌， 人体组织在该项目的第一个UG 3阶段，我们将开发我们的技术， 可靠地分析任何大块人体组织。在UG 3的目标1中，我们将开发一种技术， 超高保真度的单链和双链嵌合突变（取代、插入、缺失、结构突变） 变量和retroelements）。在UG 3的目标2中，我们将使用单分子的机器学习模型 聚合酶动力学检测不同类型的单链DNA损伤和修饰。重要的是， HiDEF-seq将在一次检测中同时检测所有这些事件。在第二个UH 3阶段 在该项目中，我们将与SMaHT网络密切合作，以验证和扩展 该技术，使它可以分析整个收集的SMaHT组织样本。在UH 3的目标1中，我们将 完全自动化HiDEF-seq的实验室组件，以创建数百个测序文库 每天的样品。在UH 3的目标2中，我们将扩展我们技术的计算管道， 分析了数千个样本。在整个项目中，我们将与SMaHT网络合作， 标准化和推广技术。HiDEF-seq实现超高保真测序， 单链DNA突变和损伤将使从根本上新型的嵌合突变研究， 将解开DNA突变，修复和复制的相互关联的过程。它还将使 对实验室处理DNA过程中产生的人工制品来源进行了系统的剖析。此外，它将 揭示了外源诱变剂的瞬时效应和时间动态，具有广泛的意义， 环境健康和发现减少或增加我们基因组突变率的因素。