Nuclease Probe Mediated Sequencing

描述（由申请人提供）：测序对于鉴定可能调节细胞功能和疾病、使人易患某些疾病或警告不良药物代谢的基因组DNA差异非常有效。它为鉴定基因表达差异提供了基础，尽管这样的应用受到限制并且存在问题，因为每个表达的基因可以从每个细胞的单个拷贝变化到成千上万个拷贝。此外，其应用于福尔马林固定石蜡包埋组织（FFPE）是相当具有挑战性的。数以百万计的此类样本沿着相应的治疗方式和已知的临床结果在临床中心和医院存档。数百万个这样的样本也已经从体内安全性、代谢和疾病动物模型研究中存档。此外，测序对于常规实验的效用（其中目前使用基于阵列的测量）受到成本的限制，并且还可能受到测序数据的准确性、灵敏度和再现性的限制。该第一阶段提案开发并验证了一种用于解决这些挑战的新方法“核酸酶探针介导测序”，提供了用于将测序集中到感兴趣的基因上的候选捕获方法的变体，并解决了基于阵列的方法的问题。所提出的方法使用仅裂解测定和HTG的定量核酸酶保护测定（qNPA”）的变体或连接/核酸外切酶产生的挂锁探针以产生用于合成测序的DNA探针，与基因标记和实验标记方法组合以允许合并以提供成本降低和更高的样品通量。 公共卫生相关性：该I期项目将解决使用测序测量基因表达的挑战。所提出的方法将使研究人员能够使用存档的固定组织样本。它还将降低每个样品的成本，并增加每次测序运行测试的样品数量，同时潜在地增加测量的准确性、动态范围和再现性，扩展测序测量基因表达的效用，并提供比目前由基于高密度阵列的测量提供的更具成本效益和精确的平台。

DESCRIPTION (provided by applicant): Sequencing is very powerful for identifying differences in genomic DNA that may regulate cell function and diseases, pre-dispose persons to certain diseases, or warn of adverse drug metabolism. It provides a basis for identifying differences in gene expression, though such applications have been limited and are problematic because each expressed gene can vary from a single copy per cell to 10's of thousands of copies. Further, its application to formalin fixed paraffin-embedded tissue (FFPE) is quite challenging. Millions of such samples, along with the corresponding treatment modalities and known clinical outcomes, are archived at clinical centers and hospitals. Millions of such samples have also been archived from in vivo studies of safety, metabolism, and animal models of disease. In addition, the utility of sequencing for routine experiments where array-based measurements are used today is limited by cost, and might also be limited by accuracy, sensitivity, and reproducibility of the sequencing data. This Phase I proposal develops and validates a new approach for addressing these challenges, "Nuclease Probe Mediated Sequencing," provides a variation of candidate capture methods used to focus sequencing onto genes of interest, and addresses issues with array-based methods. The method proposed uses a lysis-only assay and either a variation of HTG's quantitative Nuclease Protection Assay (qNPA") or ligation/exonuclease generated Padlock probes to generate DNA probes for sequencing by synthesis, combined with gene tagging and experiment tagging methods to permit pooling to provide cost reduction and higher sample throughput. PUBLIC HEALTH RELEVANCE: This Phase I project will address challenges to using sequencing for measuring gene expression. The proposed method will enable researchers to use archived fixed tissue samples. It will also lower the cost per sample and increase the number of samples tested per sequencing run, while potentially increasing the accuracy, dynamic range and reproducibility of measurement, expanding the utility of sequencing to measure gene expression and providing a more cost effective and precise platform than currently provided by high density array-based measurement.