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 在每个健康细胞中，体细胞镶嵌突变会随着时间的流逝而积累，但检测到它们需要专业 具有极低错误率的测序技术。但是，所有目前用于分析马赛克的技术 突变需要扩增DNA，该DNA引入了单链DNA伪像。因此，甚至是 最高的富达技术只有在镶嵌突变存在于两个链中时才能检测到它们 原始DNA，但他们无法检测到其起源的单链突变和损害。这里， 我们开发了一种可以直接对DNA分子进行测序而不会在超高时进行任何扩增的技术 富达，使得DNA分子的两个链中的一根突变和损伤可以是 第一次检测到。它通过显着提高单分子DNA的准确性来实现这一目标 顺序，此外，它利用可用于研究基因组区域的长读数 使用简短读数的所有先前的高保真镶嵌突变技术都无法访问。我们的技术， 称为发夹双工增强的富达测序（hidef-seq），将作为SMAHT的一部分开发 网络，我们将在项目的各个阶段与网络密切协调，以确保 在网络中创建全面的躯体镶嵌目录的目标极大地做出了贡献 人体组织。在该项目的第一个UG3阶段，我们将开发我们的技术以成本效益和 可靠地介绍了任何散装的人体组织。在UG3的AIM 1中，我们将开发技术来介绍所有类别的所有类别 超高保真度（取代，插入，缺失，结构性）时单链和双链镶嵌突变 变体和重新元素）。在UG3的AIM 2中，我们将使用单分子的机器学习模型 聚合酶动力学检测各种类型的单链DNA损伤和修饰。重要的是， hidef-seq将仅在一个测定中就可以实现所有这些事件的检测。在第二个UH3阶段 该项目，我们将与SMAHT网络密切合作，以验证和扩展 该技术可以介绍整个SMAHT组织样品的集合。在UH3的AIM 1中，我们将 完全自动化Hidef-Seq的实验室组件，以创建数百个测序库 每天样品。在UH3的AIM 2中，我们将扩展技术的计算管道以进行快速 分析数千个样本。通过这个项目，我们将与SMAHT网络合作以验证， 标准化并传播技术。 hidef-seq的超高忠诚测序的成就 单链DNA突变和损伤将使从根本上实现新型的镶嵌突变研究，使得 将解散DNA突变，修复和复制的相互关联过程。它也将启用 由DNA实验室加工引起的伪像的系统解剖。此外，它将 揭示外源诱变剂的瞬时作用和临时动力学，对 环境健康和发现降低或增加基因组突变速率的因素。