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

摘要 下一代测序（NGS）已成为临床肿瘤学实践中日益不可或缺的一部分， 在那里，其可扩展地检查数百到数千个目标的能力现在常规地能够识别 支持精准医疗实践的预后和治疗可操作标志物。有 许多应用中，检测和定量癌症相关的基因型在超 低水平（<1/10，000或更高），例如识别肿瘤中的耐药突变，检测残留的 治疗后的癌细胞，或早期癌症检测。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（约1/100 bp），低于此值，无法区分真正的生物变异， 噪声已经提出了各种方法来通过允许NGS序列的纠错来绕过这个问题 读取，但是这样的技术需要在高深度处冗余地测序单个模板分子， 可以产生纠错的共有序列。因此，这些方法需要大量的 测序能力，是昂贵的，并在数量的标本和基因组目标，可以是有限的。 考察因此，它们在临床应用中的应用很少。仍然有一个未得到满足的需要， 精确的测序方法，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用途。我们最近开发了一种新的实验范式，称为“链接 双链体测序”，解决了这些局限性。在我们的方法中，我们将两条DNA链连接起来， 将初始模板片段转化为单个共价连接的分子。双工器的纠错可以是 通过比较来自两条连接链的单独读段来进行，从而消除了对冗余读段的需要。 模板分子的测序。这项创新技术提供了强大的纠错能力和可扩展性， 成本效益，效率和定量精度，并与低到中输出短读兼容 测序平台（即，Illumina）已经在广泛的临床使用。在我们的第一个目标中，我们将开发 支持连锁双链体测序的工作流程，将开发支持性生物信息学分析管道， 将使用新鲜的和福尔马林固定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，我们将开发用于靶向富集的基因或变体的方案， 他对连锁双链体测序感兴趣，并将使用各种临床材料评估性能。这 工作将提供信息和可交付成果，对癌症有直接，直接和变革性的益处 通过大幅提高肿瘤测序检测的质量，同时向患者灌输 增强的诊断能力，用于超灵敏地检测与以下相关的癌症相关突变： 疾病的出现、复发和治疗抗性。我们的目标是制造超灵敏， 纠错测序如此便宜和简单，它将被用作标准操作， 用于NGS临床肿瘤学测定和癌症研究的程序。

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.