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.

项目概要/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技术 错误率极低的测序技术 然而，目前所有的分析马赛克技术。 突变需要 DNA 扩增，这会引入单链 DNA 伪影，因此，即使是 最高保真度技术只能检测嵌合突变，当它们存在于两条链中时 原始 DNA，但它们无法检测到它们起源的单链突变和损伤。 我们开发了一种技术，可以直接对 DNA 分子进行测序，无需任何超高扩增 保真度，使得仅存在于 DNA 分子两条链之一的突变和损伤可以被 它通过显着提高单分子 DNA 的准确性来首次实现这一目标。 测序，此外，它利用长读长，可用于研究基因组中的区域 所有先前使用我们的技术的高保真嵌合突变技术都无法实现这一点， 称为发夹双工增强保真测序 (HiDEF-seq)，将作为 SMaHT 的一部分进行开发 网络，我们将在项目的各个阶段与网络密切协调，以确保 为网络创建体细胞嵌合体综合目录的目标做出了重大贡献 在该项目的第一个 UG3 阶段，我们将开发我们的技术，以实现成本效益和成本效益。 可靠地分析任何大块人体组织 在 UG3 的目标 1 中，我们将开发分析所有类别的技术。 超高保真度的单链和双链嵌合突变（替换、插入、删除、结构 在UG3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。 HiDEF-seq 将在第二个 UH3 检测阶段同时实现所有这些事件的检测。 在该项目中，我们将与 SMaHT 网络紧密合作，以验证和扩展 SMaHT 的吞吐量 该技术使其能够分析整个 SMaHT 组织样本集合 在 UH3 的目标 1 中，我们将。 HiDEF-seq 的实验室组件完全自动化，可创建数百个测序库 在 UH3 的目标 2 中，我们将扩展我们技术的计算管道以实现快速计算。 在整个项目中，我们将与 SMaHT 网络合作来验证、 标准化并传播 HiDEF-seq 超高保真测序技术的成果。 单链 DNA 突变和损伤将从根本上实现新型嵌合突变研究 将解开 DNA 突变、修复和复制的相互关联过程。 此外，它将对实验室 DNA 处理产生的文物来源进行系统剖析。 揭示外源诱变剂的瞬时效应和时间动态，对 环境健康以及减少或增加基因组突变率的因素的发现。