Error-suppressed whole genome sequencing for genotoxicant-induced structural variant detection

用于基因毒物诱导的结构变异检测的错误抑制全基因组测序

• 批准号: 10590370
• 负责人: THOMAS EDWARD WILSON
• 金额: $ 41.84万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-14 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10590370
• 关键词: Address; Adopted; Affect; Alkylating Agents; Base Pairing; Basic Science; Biological Assay; Bone Marrow Purging; Bone Marrow Transplantation; Brain; Cell Line; Cells; Chemicals; Chromosome Breakage; Comet Assay; Constitution; Constitutional; Copy Number Polymorphism; DNA; DNA Repair; Data; Detection; Disease; Double Strand Break Repair; Ensure; Environmental Exposure; Epigenetic Process; Equilibrium; Evaluation; Event; Evolution; Exposure to; Frequencies; Gene Fusion; Genes; Genetic Diseases; Genetic Predisposition to Disease; Genome; Genomics; Goals; Hazard Identification; Heterogeneity; High-Throughput Nucleotide Sequencing; Human; Human Genetics; Inherited; Lead; Libraries; Ligation; Malignant Neoplasms; Materials Testing; Methods; Micronucleus Tests; Modeling; Molecular; Morphologic artifacts; Mosaicism; Mus; Mutagenesis; Mutagens; Mutation; Mutation Detection; Nature; Noise; Patients; Performance; Pilot Projects; Process; Protocols documentation; Risk; Risk Factors; Signal Transduction; Single Nucleotide Polymorphism; Specificity; Specimen; Testing; Tissues; Toxic effect; Toxicogenetics; Variant; Work; Yeasts; artemis; clastogen; cost effectiveness; design; disorder risk; follow-up; genome sciences; genome sequencing; genome-wide; genotoxicity; hazard; in vivo; inter-individual variation; interest; mosaic; mutant; population survey; prevent; response; screening; success; targeted nucleases; tool; translational potential; variant detection; whole genome

Project Summary Structural variants (SVs) are an important class of human genetic alteration that creates large changes in genomic content in single mutational events. Copy number variants (CNVs) change the representation of thousands to millions of DNA base pairs, potentially including multiple genes, whereas copy number neutral inversions and translocations lead to gene fusions and dysregulation of epigenetic control mechanisms. SVs and CNVs are critical drivers of cancer, a primary mechanism of constitutional germline genetic disease, and are increasingly appreciated as an ongoing form of somatic mosaicism affecting the function of tissues. SVs are subject to the same classes of risk factors as single-nucleotide variants (SNVs). These factors, which lead to inter-individual variation in lifetime mutation burden and therefore disease risk, include inherited predispositions based on DNA repair capacity and environmental exposures to mutagenic genotoxicants. Genotoxicants that induce disruption or breakage of chromosomes are known as clastogens. Importantly, assays commonly used to identify clastogens or double-strand break (DSB)-inducing genotoxicants are non-specific and fail to reveal whether specific agents lead to new, stable SV junctions in cells and, if so, what the nature and distribution of those junctions is. There is a large need for assays that can efficiently reveal SV mutagens via definitive, positive readouts of breakpoint junctions, the molecular endpoint of interest to toxicologists. Such an assay would be highly impactful in the genome sciences as a way of surveying populations of cells for mosaic SV mutations, including in studies of hazard identification and evaluation, as well as in basic science studies of cancer evolution and heterogeneity, tissue somatic mosaicism, and DSB repair mechanisms. SNVs have further demonstrated the great value of error-minimized, high-throughput sequencing for mutation detection and genotoxicant characterization. Led by Duplex Sequencing, such approaches reveal baseline and induced SNV frequencies with exquisite sensitivity by maximizing signal-to-noise ratios. However, prior work on error-minimized sequencing has been largely limited to SNVs. Our data confirm that the approaches do not address the distinct error mechanisms that lead to false SV detections. Our rationale is that an error-minimized sequencing method designed to address the processes that give rise to SV artifacts will be as impactful for SVs as Duplex Sequencing has been for SNVs. This R21 project will devise, develop, and validate such a method and begin to apply it to important genetic toxicology paradigms, with the following aims: (1) Develop a robust svWGS protocol for error-suppressed, non-targeted SV junction sequencing; and (2) Validate svWGS using well-controlled, high-value case examples with translational potential. Our long-term objective is to apply svWGS in detailed characterizations of emerging candidate human clastogens.

项目摘要 结构变异（SVS）是一类重要的人类遗传改变，会在 单个突变事件中的基因组含量。拷贝数变体（CNV）更改的表示形式 数千至数百万的DNA碱基对，可能包括多个基因，而拷贝数中性 反转和易位会导致基因融合和表观遗传控制机制的失调。 SVS和 CNV是癌症的关键驱动因素，癌症是宪法种系遗传疾病的主要机制，是 越来越多地成为影响组织功能的一种持续形式的体细胞镶嵌形式。 SV与单核苷酸变体（SNV）相同的危险因素。这些因素， 这导致终生突变负担和疾病风险的个体差异，包括遗传 基于DNA修复能力和对诱变基因毒性剂的环境暴露的易感性。 诱导破坏或破裂染色体的遗传毒性称为clastogens。重要的是，测定法 通常用于鉴定锁骨剂或双链断裂（DSB）诱导遗传毒性剂是非特异性的，并且 无法揭示特定的代理是否导致细胞中新的稳定的SV连接，如果是的，则性质和性质是什么 这些连接的分布是。对于可以通过 断点连接的确定，积极的读数，毒理学家感兴趣的分子终点。这样的 在基因组科学中，测定将对马赛克的细胞种群进行高度影响 SV突变，包括危害识别和评估的研究，以及 癌症进化和异质性，组织体细胞镶嵌和DSB修复机制。 SNV进一步证明了误差最少的高通量测序的巨大价值 突变检测和遗传毒性表征。在复式测序的领导下，这种方法揭示了 基线和诱导的SNV频率具有最大的信号噪声比，具有精致的灵敏度。然而， 关于错误最定测序的先前工作在很大程度上限于SNV。我们的数据证实了 方法不能解决导致错误SV检测的独特错误机制。我们的理由是 旨在解决引起SV工件的过程的错误最小化测序方法将是 对SVS的影响与双链测序对SNV的影响已经是SNV。这个R21项目将设计，开发和 验证这种方法，并开始将其应用于重要的遗传毒理学范例，以下目的： （1）为错误抑制的，非目标的SV连接测序开发可靠的SVWGS协议； （2） 使用具有转化潜力的高价值案例示例来验证SVWG。我们的长期 目的是将SVWG应用于新兴候选人类clastogens的详细特征。