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

项目摘要/摘要 随着时间的推移，体细胞花叶突变会在每个健康细胞中积累，但检测它们需要专门的 测序技术具有极低的错误率。然而，目前所有用于剖析马赛克的技术 突变需要DNA扩增，这就引入了单链DNA伪像。因此，即使是 最高保真度的技术只能在马赛克突变存在于两条链上时才能检测到它们 原始DNA，但它们无法检测到它们起源的单链突变和损伤。这里, 我们开发了一种技术，可以在超高频率下直接对DNA分子进行测序，而不需要任何放大 保真度，即DNA分子的两条链中只有一条存在突变和损伤 首次检测到。它通过显著提高单分子DNA的准确性来实现这一点 测序，而且，它利用了可以用来研究基因组区域的长读数 不能被所有使用短读的现有高保真镶嵌突变技术所访问。我们的技术， 名为发夹双工增强型保真度测序(HiDEF-seq)，将作为SMAT的一部分开发 我们将在项目的所有阶段与该网络密切协调，以确保 为该网络的目标做出了重大贡献，即创建一个全面的 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，以具有成本效益和 可靠地分析任何块状人体组织。在UG3的目标1中，我们将开发技术来分析所有类别的 超高保真的单链和双链镶嵌突变(替换、插入、缺失、结构 变异体和反转录元件)。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。重要的是 HiDEF-SEQ将在一次检测中实现对所有这些事件的同时检测。在第二个UH3阶段 作为该项目的一部分，我们将与SMAHT网络密切合作，以验证和扩展 这项技术这样它就可以分析整个SMAHTT组织样本的收集。在UH3的目标1中，我们将 完全自动化HiDEF-seq的实验室组件，以创建数百个测序库 每天的样本数量。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 对数千个样本进行了分析。在整个项目中，我们将与SMATH网络合作验证， 规范、推广这项技术。HiDEF-SEQ实现超高保真测序 单链DNA突变和损伤将从根本上实现新型马赛克突变研究， 将解开DNA突变、修复和复制的相互关联的过程。它还将启用 对实验室DNA处理产生的人工制品来源进行系统剖析。此外，它还将 揭示外源诱变剂的瞬时效应和时间动力学，具有广泛的意义 环境健康和发现降低或提高我们基因组突变速度的因素。

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.