SlipChip for Target Enrichment for Next-Gen Sequencing by Multiplexed PCR

描述（由申请人提供）：在过去的30年里，桑格测序已经能够确定一小段DNA上的核苷酸碱基序列，这项技术的最高成就是耗资数亿美元完成了人类基因组的完整阐明。近年来，“下一代”测序技术已经发展到能够并行处理数百万个DNA模板。与最初人类基因组计划的天文数字成本相比，目前使用下一代测序技术对一个完整的人类基因组进行测序的成本目前不到5万美元。然而，对于大多数需要对基因组重点区域进行深度测序的科学研究或临床研究来说，即使这种相对较低的成本仍然太高。因此，有针对性的富集策略是必要的，即只富集所需的基因组片段以供后续测序。而且，目前还没有一种简单的、个人研究或临床实验室负担得起的、结合质量控制的靶向富集解决方案。SlipChip是一项有吸引力的创新技术，可以潜在地解决这一问题。SlipChip已经应用于酶分析、免疫分析和PCR分析等复杂方案。pi 14已经证明，玻璃制造的SlipChips可以用于执行多重PCR和数字PCR。该I期提案的目标是在16个多重PCR领域建立这种创新的可行性和适用性，用于靶向富集和随后的测序，同时测试测序工作流程中上游和下游步骤的可行性。目标1)，我们将测试和验证18个多重PCR在SlipChip中使用人类DNA的鲁棒性，目标2)，我们将优化SlipChip的密度，最后，目标3)，我们将验证在提交样品之前的PCR片上质量控制是可行的。20实现这些具体目标将牢固地确立SlipChip技术作为下一代多重PCR测序靶向富集的样品处理工具的可行性和可行性，并将22降低该领域二期工作的技术风险，其中包括在高度重要的生物医学问题背景下的测序实验。并将允许对经过验证的SlipChip 24系统进行研究，以便将其完全集成到现有下一代测序工具（特别是25 Illumina GA IIe）的工作流程中。

DESCRIPTION (provided by applicant): 1. For the past 30 years, Sanger sequencing has enabled the determination of a sequence of nucleotide 2 bases on a small segment of DNA, with the crowning achievement of the technology being the complete 3 elucidation of the human genome at a cost of several hundreds of millions of dollars. Recently, "next- 4 generation" sequencing technologies have been developed capable of processing millions of DNA templates in 5 parallel. Compared to the astronomical cost of the original human genome project, the current cost to 6 sequence a complete human genome using next-generation sequencing technology is currently under 7 $50,000. However, even this relatively lower cost is still too high for the majority of scientific inquiry or clinical 8 studies that require deep sequencing of focused regions of the genome. Thus, targeted enrichment strategies 9 that enrich only desired segments of the genome for subsequent sequencing are necessary. And, there is 10 currently no solution for targeted enrichment exists that is simple, affordable to individual research or clinical 11 laboratories, and that incorporates quality control. 12 SlipChip is an attractive and innovative technology that can potentially solve this problem. SlipChip has 13 already been applied to complex protocols such as enzyme assays, immunoassays, and PCR assays. The PIs 14 have demonstrated that SlipChips fabricated in glass can be used to perform multiplexed PCR and digital PCR. 15 The goal of this Phase I proposal is to establish the feasibility and applicability of this innovation in the area of 16 multiplexed PCR for targeted enrichment and subsequent sequencing, while testing the feasibility of upstream 17 and downstream steps in the sequencing workflow: from Aim 1) where we will test and verify the robustness of 18 multiplexed PCR in the SlipChip using human DNA, Aim 2) where we will optimize the density of SlipChip and 19 finally, Aim 3) where We will verify that on-chip quality control of PCR before committing a sample is feasible. 20 Reaching these specific aims would firmly establish feasibility and viability of SlipChip technology as a 21 sample processing tool for targeted enrichment for next generation sequencing by multiplexed PCR, and would 22 reduce the technical risk of Phase II work in this area, which would include sequencing experiments in the 23 context of biomedical problems of high significance, and will allow for the study of the validated SlipChip 24 system for complete integration in the workflow of an existing next-generation sequencing tool, specifically the 25 Illumina GA IIe. PUBLIC HEALTH RELEVANCE: 1 Technologies for the targeted enrichment of specific genomic regions are urgently needed to bring the 2 power of next-generation sequencing to fundamental and applied biomedical research. However, no solution 3 for targeted enrichment exists that is simple and affordable to individual research or clinical laboratories. This 4 proposal describes a SlipChip technology to address this unmet need.