Nuclease Probe Mediated Sequencing

该第二阶段提案将完成创新测序平台的开发 (NPSeq)基于第一阶段论证的可行性。该平台使用核酸酶保护 实验与通用聚合酶链式反应相结合，以产生靶向测序文库。仅限裂解 协议意味着不需要提取RNA或DNA，也不需要逆转录RNA。 因此，固定组织的测序与非固定组织的测序一样灵敏和准确 组织，解决药物发现、转化研究和诊断方面尚未得到满足的需求 并提供大量存档的临床固定组织，患者对这些组织的结果 都是已知的，但很难通过RNAseq进行测序。使用NPSeq的性能比较 FFPE与匹配的非固定组织的结果比来自RNA的RNAseq测量结果更好 从相同的样本中提取。NPSeq是定量的、灵敏的和高度可重复性的 在变异系数为3%的情况下，无论是10个，还是多达1000个或更多的基因都可以被测量。因此， 相同的化验/平台可用于生物标记物鉴定以及验证， 验证，以及最终的重点应用只测量几个基因，无论它是一种药物 发现试验或体外诊断(IVD)。我们用一种化验证明了这种能力 在来自miRBase 18的1,942个miRNA的整个miRNome中，超过了第一阶段的目标。 我们还证明了mRNA和miRNA可以在同一个测试中被测量，这是 重要提示：虽然从FFPE中可以比从mRNA中更准确地测量到miRNA， 需要测量管家的mRNA基因，因为患病的、非坏死性的 每个样本中的组织都可能是高度可变的。因此，双重测量的能力 MRNA/miRNA可以改善数据，并利用该方法固有的(3%)重复性。在……里面 在第二阶段，我们将优化、验证和验证mRNA和miRNA商业协议， 并开发程序来测量作为药物靶点的mrna突变，例如基因融合 并表达SNPs(ESNPs)。我们将生成更新的、完整的miRNome NPSeq分析 (miRbase 19+试验)。我们还将为整个转录组确定可用的探针，并 建立一个基因-疾病关联的数据库；这两个工具结合在一起将允许快速 开发可定制的化验方法。这些工具将用于生成几种分析 在第二阶段。然后我们将使用miRBase 19+和 几种信使核糖核酸分析-用细胞系和匹配的冷冻和固定的数据产生数据 临床组织以证明这些NPSeq分析的实用性并进行试剂盒 开发和产品发布。我们希望化验结果能为调查人员提供一条通道 为其研究或AS发现、验证、验证和应用生物标记物 诊断化验。

This Phase II proposal will complete the development of an innovative sequencing platform (NPSeq) based on feasibility demonstrated in Phase I. This platform uses a nuclease protection assay combined with universal PCR to produce targeted sequencing libraries. The lysis-only protocol means there is no need to extract RNA or DNA or to reverse transcribe RNA. Consequently, sequencing from fixed tissue is as sensitive and accurate as sequencing unfixed tissue, addressing an unmet need for drug discovery, translational research, and diagnostics and making available vast amounts of archived clinical fixed tissue for which patient outcomes are known but are difficult to sequence via RNAseq. Comparative performance of NPSeq using FFPE vs. matched unfixed tissue results were better than RNAseq measurements from RNA extracted from the same samples. NPSeq is quantitative, sensitive, and highly reproducible with CV's of 3%, whether 10 or up to a thousand or more genes are measured. As a result, the same assay/platform can be used for biomarker identification as well as for verification, validation, and the final focused application measuring just a few genes, whether it is a drug discovery assay or an in vitro diagnostic (IVD). We demonstrated this capability with an assay of the whole miRnome of 1,942 miRNA from miRbase 18, overachieving on the Phase I Aims. We also demonstrated that mRNA and miRNA can be measured in the same assay, which is important: while miRNA can be measured from FFPE with greater success than mRNA, housekeeper mRNA genes need to be measured because the amount of diseased, non-necrotic tissue in each sample can be highly variable. Thus, the capability for dual measure of mRNA/miRNA can improve data and exploit the intrinsic (3%) reproducibility of the method. In this Phase II we will optimize, verify and validate the mRNA and miRNA commercial protocols, and develop protocols to measure mRNA mutations that are drug targets, such as gene fusions and expressed SNPs (eSNPs). We will generate an updated, whole-miRNome NPSeq assay (the miRbase19+ assay). We will also identify usable probes for the entire transcriptome and establish a database of gene-disease associations; these two tools together will allow for rapid development of customizable assays. These tools will be used to generate several assays during Phase II. We will then go through product development with the miRbase 19+ and several of the mRNA assays - generating data with cell lines and matched frozen and fixed clinical tissue to demonstrate the utility of these NPSeq assays and proceeding to kit development and product release. We expect that assays will provide investigators a pipeline for discovery, verification, validation, and application of biomarkers for their research or as diagnostic assays.