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

摘要 下一代测序(NGS)已日益成为临床肿瘤学实践中不可或缺的一部分， 现在，它能够可扩展地检查数百到数千个目标，从而能够定期识别 支持精确医学实践的预后和治疗可操作标记物。确实有 在许多应用中，检测和定量检测与癌症相关的基因类型将是有用的 低水平(每10,000人中有1人或更多)，如识别肿瘤中的耐药突变、检测残留 癌细胞治疗后，或早期发现癌症。然而，标准的NGS技术受到以下因素的阻碍 相对较高的错误率(~1/100个基点)，低于此错误率无法区分真正的生物变异 噪音。已经提出了通过允许对NGS序列进行纠错来绕过该问题的各种方法 但这种技术需要在高深度对单个模板分子进行冗余测序 可以产生纠错的共识序列。因此，这些方法需要大量的 测序能力，是昂贵的，并且在样本和基因组靶标的数量上受到限制 检查过了。因此，它们在临床应用中几乎没有被吸收。仍有高度的需求未得到满足 准确的测序方法，具有成本效益，可扩展，并允许询问足够的基因靶点 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“关联” 双链测序“，它解决了这些限制。在我们的方法中，我们将来自 最初的模板片段变成单个共价连接的分子。双工的纠错可以是 通过比较来自两个连接的链的单独读取来执行，从而消除对冗余的需求 模板分子的测序。该创新技术提供具有可伸缩性的健壮纠错， 性价比高、效率高、定量精度高，并与中低输出短读兼容 已经在临床上广泛使用的测序平台(如Illumina)。在我们的首要目标中，我们将发展 支持链接双链测序的工作流程，将开发支持性生物信息分析管道，以及 将使用新鲜的和固定了福尔马林的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，我们将开发有针对性的浓缩基因或变种的方案 对链接的双链测序感兴趣，并将使用各种临床材料评估性能。这 工作将为癌症提供直接、直接和变革性的信息和交付成果 通过显著提高肿瘤测序分析的质量，同时向患者灌输 增强了对与以下相关的癌症相关突变的超灵敏检测的诊断能力 常规临床实践中的疾病出现、复发和治疗抵抗。我们的目标是让超级敏感， 纠错后的测序如此廉价和直接，将被用作标准操作 NGS临床肿瘤学分析和癌症研究程序。

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.