Nuclease Probe Mediated Sequencing

描述(由申请人提供)：测序对于识别基因组DNA的差异非常有效，这些差异可能调节细胞功能和疾病，使人易患某些疾病，或警告药物的不良代谢。它为识别基因表达的差异提供了基础，尽管这种应用一直受到限制，而且存在问题，因为每个表达的基因可能从每个细胞一个拷贝到10‘S数千个拷贝不等。此外，将其应用于福尔马林固定的石蜡包埋组织(FFPE)是相当具有挑战性的。数以百万计的此类样本以及相应的治疗方式和已知的临床结果被存档在临床中心和医院。数以百万计的此类样本也是从安全性、新陈代谢和疾病动物模型的体内研究中存档的。此外，今天使用基于阵列的测量的常规实验的测序的实用性受到成本的限制，并且可能还受到测序数据的准确性、灵敏度和重复性的限制。这项第一阶段的提案开发并验证了一种解决这些挑战的新方法，即“核酸酶探针介导的测序”，提供了一种候选捕获方法的变体，用于将测序集中在感兴趣的基因上，并解决了基于阵列的方法的问题。建议的方法使用仅裂解分析和HTG定量核酸酶保护分析(qNPA“)的变体或连接/核酸外切酶产生的挂锁探针来生成DNA探针用于合成测序，结合基因标记和实验标记方法允许汇集以提供更低的成本和更高的样本吞吐量。 公共卫生相关性：这个第一阶段项目将解决使用测序来测量基因表达的挑战。拟议的方法将使研究人员能够使用存档的固定组织样本。它还将降低每个样本的成本，增加每次测序运行测试的样本数量，同时潜在地提高测量的准确性、动态范围和重复性，扩大测序用于测量基因表达的用途，并提供比目前基于高密度阵列的测量提供的更具成本效益和更精确的平台。

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 Millions of such samples have also been archived from in vivo studies of safety, metabolism, and animal models of disease.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.