Efficient, cost-effective, and ultrasensitive sequencing of somatic mutations

高效、经济且超灵敏的体细胞突变测序

基本信息

批准号：
10488391
负责人：
Stephen J Salipante
金额：
$ 18.17万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-08 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10488391
关键词：
Accreditation Address Adoption Alleles Base Sequence Bioinformatics Biological Biological Assay Bypass CLIA certified Cancer Detection Cancer Patient Cancer cell line Characteristics Clinical Clinical Oncology Clinical Research Community Clinical Oncology Program Consensus Consensus Sequence DNA DNA sequencing Detection Development Diagnostic Disease Drug resistance Event Exhibits Formalin Frequencies Fresh Tissue Gene Mutation Gene Targeting Genes Genome Genomics Genotype Goals Individual Label Laboratories Laboratory Research Libraries Ligation Link Low Prevalence Malignant Neoplasms Measures Mediating Methodology Methods Molecular Monitor Monitoring for Recurrence Mutation Neoplasms Noise Nucleotides Oncology Output Performance Procedures Prognosis Property Protocols documentation Recurrence Relapse Residual Cancers Residual state Resistance Sampling Screening for cancer Sensitivity and Specificity Somatic Mutation Specimen Techniques Technology Therapeutic Tissues Transposase Validation Variant Work analysis pipeline anticancer research bioinformatics pipeline bioinformatics tool cancer cell cancer therapy cell free DNA clinical diagnosis clinical diagnostics clinical material clinical practice clinically actionable clinically relevant cost cost effective cost effectiveness data analysis pipeline design diagnostic value exome improved innovative technologies insertion/deletion mutation interest mutant neoplastic cell next generation next generation sequencing novel precision medicine prognostic rare cancer research clinical testing research study resistance mutation screening sequencing platform tumor uptake whole genome

项目摘要

ABSTRACT Next-generation sequencing (NGS) has become increasingly integral to the practice of clinical oncology, where its ability to scalably examine hundreds to thousands of targets now routinely enables identification of prognostic and therapeutically actionable markers that support the practice of precision medicine. There are many applications for which it would be useful to detect and quantitate cancer-associated genotypes at ultra- low levels (<1 in 10,000 or more), such as identifying drug-resistance mutations in tumors, detecting residual cancer cells after therapy, or early cancer detection. Nevertheless, standard NGS technologies are hampered by a relatively high error rate (~1 in 100bp), below which true biological variation cannot be distinguished from noise. Various methods have been proposed to bypass this issue by allowing error correction of NGS sequence reads, but such techniques require redundantly sequencing individual template molecules at high depth such that an error-corrected consensus sequence can be produced. As a result, those methods require a large amount of sequencing power, are costly, and are limited in the number of specimens and genomic targets that can be examined. They have consequently seen little uptake in clinical use. There remains an unmet need for highly accurate sequencing methods that are cost-effective, scalable, and allow interrogation of enough gene targets for meaningful use in clinical practice. We have recently developed a new experimental paradigm, termed “Linked Duplex Sequencing”, that addresses these limitations. In our approach, we join the two strands of DNA from an initial template fragment into a single, covalently linked molecule. Error correction of the duplex can be performed by comparing separate reads from the two linked strands, thereby eliminating the need for redundant sequencing of template molecules. This innovative technology provides robust error correction with scalability, cost-effectiveness, efficiency, and quantitative precision, and is compatible with low-to-mid output short read sequencing platforms (ie, Illumina) that are already in widespread clinical use. In our first Aim, we will develop workflows to support Linked Duplex Sequencing, will develop supportive bioinformatic analysis pipelines, and will characterize the cardinal performance metrics of the approach using fresh and formalin-fixed reference material. In our second Aim, we will develop protocols for the targeted enrichment of genes or variants of interest for Linked Duplex Sequencing, and will evaluate performance using a variety of clinical materials. This work will provide information and deliverables with immediate, direct, and transformative benefit to cancer patients by substantially improving the quality of oncology sequencing assays while imbuing them with enhanced diagnostic capabilities for the ultrasensitive detection of cancer associated mutations relevant to disease emergence, relapse, and therapy resistance in routine clinical practice. Our goal is to make ultrasensitive, error corrected sequencing so inexpensive and straightforward that it will be used as standard operating procedure for NGS clinical oncology assays and cancer research studies.

抽象的下一代测序（NGS）已成为临床肿瘤学实践越来越不可或缺的，现在，其能够可靠地检查数百到数千个目标的能力现在常规地识别支持精密医学实践的预后和热可操作的标记。有在超级 - 检测和定量与癌症相关的基因型上的许多应用将是有用的低水平（<10,000或更多），例如鉴定肿瘤中的药物抗性突变，检测残留治疗后癌细胞或早期癌症检测。然而，标准NGS技术受到阻碍相对较高的错误率（100bp中的1个），在此下面无法区分真正的生物学变化噪音。已经提出了各种方法来绕过此问题，允许对NGS序列进行错误校正读取，但是这种技术需要在高深度上进行冗余测序单个模板分子。可以产生错误校正的共识序列。结果，这些方法需要大量测序能力的昂贵，并且在可以是的标本和基因组靶标数量上受到限制检查。因此，他们看到临床用途几乎没有吸收。对高度的需求仍未满足具有成本效益，可扩展性的准确测序方法，并允许对足够的基因靶标进行询问在临床实践中有意义的使用。我们最近开发了一种新的实验范式，称为“链接双工测序”，解决这些局限性。在我们的方法中，我们从初始模板片段成一个共价连接的分子。双工的错误校正可以是通过比较来自两个链接链的单独读数来执行，从而消除了对冗余的需求模板分子的测序。这种创新的技术可提供可伸缩性的鲁棒性校正性，成本效益，效率和定量精确度，与低到中的输出兼容简短读取测序平台（IE，Illumina）已经处于临床范围内。在我们的第一个目标中，我们将发展支持链接的双工测序的工作流程，将开发支持性的生物信息学分析管道，并将使用新鲜和福尔马林固定参考来表征该方法的主要性能指标材料。在我们的第二个目标中，我们将开发针对基因或变体的靶向富集的协议对链接的双链测序的兴趣，并将使用各种临床材料评估性能。这工作将提供立即，直接和变革性的好处的信息和可交付成果，以取消通过实质上提高肿瘤学测序测定的质量，同时使患者融入增强了与癌症相关突变的超敏感检测的诊断能力常规临床实践中的疾病紧急，继电器和治疗性。我们的目标是使超敏错误纠正的测序如此便宜且直接，以至于将其用作标准操作 NGS临床肿瘤学分析和癌症研究的程序。