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

描述(申请人提供)：1.在过去的30年里，桑格测序已经能够根据一小段DNA来确定核苷酸2的碱基序列，这项技术的最大成就是花费数亿美元完成了人类基因组的阐明。最近，“下一代”测序技术已经发展起来，能够5次并行处理数百万个DNA模板。与原始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50,000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重点区域进行深度测序的科学研究或临床研究来说仍然太高了。因此，有必要制定针对性的富集化策略9，仅对基因组的所需片段进行富集化，以便随后进行测序。而且，目前还没有针对靶向浓缩的解决方案，这种解决方案简单，对单个研究或临床实验室来说负担得起，并纳入了质量控制。12 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已经被应用于复杂的程序，如酶分析、免疫分析和聚合酶链式反应分析。PI 14已经证明，在玻璃中制造的SlipChips可以用于执行多重PCR和数字PCR。15本第一阶段提案的目标是确定这项创新在16个多重PCR用于靶向浓缩和后续测序方面的可行性和适用性，同时测试测序工作流程中上游17个步骤和下游步骤的可行性：从目标1)我们将使用人类DNA在芯片中测试和验证18个多路PCR的稳健性，目标2)我们将优化切片芯片的密度，最后，目标3)在提交样本之前，我们将验证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SlipChip技术作为一种样品处理工具的可行性和可行性，用于通过多路聚合酶链式反应进行下一代测序的靶向浓缩，22并将减少这一领域第二阶段工作的技术风险，这将包括在具有高度重要性的生物医学问题的背景下进行测序实验，并将允许对经过验证的SlipChip 24系统进行研究，以便完全整合到现有下一代测序工具，特别是Illumina GA IIe的工作流程中。 公共卫生相关性：1迫切需要有针对性地丰富特定基因组区域的技术，以将下一代测序的力量应用于基础和应用生物医学研究。然而，不存在简单且个别研究或临床实验室负担得起的靶向浓缩解决方案3。这份提案描述了一种SlipChip技术，以解决这一未得到满足的需求。

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.