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

描述（由申请人提供）：1。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2碱基序列，该技术的最高成就是花费数亿美元完全阐明了人类基因组。最近，已经开发出能够并行处理数百万个DNA模板的“下一代”测序技术。与最初的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，即使这种相对较低的成本对于大多数需要对基因组的重点区域进行深度测序的科学调查或临床研究来说仍然太高。因此，仅富集基因组的所需区段用于后续测序的靶向富集策略9是必要的。而且，目前还没有一种简单、个人研究或临床实验室负担得起的、并结合质量控制的靶向富集解决方案。12滑动芯片是一种有吸引力的创新技术，可以潜在地解决这个问题。滑动芯片已经被应用于复杂的方案，如酶测定，免疫测定和PCR测定。PI 14已经证明，在玻璃中制造的滑动芯片可以用于执行多重PCR和数字PCR。本第一阶段提案的目标是确定本创新在多重PCR领域的可行性和适用性，用于靶向富集和后续测序，同时测试测序工作流程中上游和下游步骤的可行性：从目标1）中，我们将测试和验证在使用人DNA的滑动芯片中18个多重PCR的稳健性，目标2）中，我们将优化滑动芯片的密度，最后，目的3）我们将验证在提交样品之前进行PCR的芯片上质量控制是可行的。20实现这些具体目标将坚定地确立滑动芯片技术作为多重PCR下一代测序靶向富集的样本处理工具的可行性和生存能力，并将减少这一领域第二阶段工作的技术风险，这将包括在高度重要的生物医学问题背景下的测序实验，并将允许研究经验证的SlipChip 24系统，以完全整合到现有的下一代测序工具，特别是25 Illumina GA IIe的工作流程中。 公共卫生相关性：1迫切需要特定基因组区域的靶向富集技术，以将下一代测序的力量带到基础和应用生物医学研究中。然而，不存在对于个体研究或临床实验室来说简单且负担得起的用于靶向富集的解决方案3。该提案描述了一种滑动芯片技术，以解决这一未满足的需求。

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.