A molecular barcoding sequencing kit for highly efficient and accurate single cel

描述（由申请人提供）：我们建议开发和商业化高灵敏度和准确性RNA测序试剂盒，适用于有限输入样品（如单细胞）中的基因表达测量。在第一阶段，我们提出了使用DNA序列标签形式的分子条形码来标记RNA的单个分子（或拷贝）以用于单细胞中的基因表达分析的概念。第一阶段项目被证明是高产的，可行性得到了证明。计数条形码提供了表达的转录物数量的绝对数字定量测量。此外，条形码被证明可用于校正PCR偏倚，这是需要DNA扩增方案的小样本中的常见挑战。我们还确定了当前单细胞RNA测序样品制备方法中0.1-3.8%总产率的低RNA表示效率，并证明了使用我们的cDNA扩增和条形码化方法，以约22.5%的显著更高的产率获得更准确的测量。这些显着的结果表明，我们的方法可以显着改善目前的技术，遭受大的损失，并有不准确的放大失真。由于基因表达水平的测量在生命科学研究和许多临床环境中都很重要，因此我们提出的产品如果成功，将大大有助于这些领域的进步。对于第二阶段，我们建议将经过验证的概念扩展到产品开发计划中，以生产高效，用户友好的单细胞RNA测序试剂盒。具体而言，我们将进行检测试剂盒开发，以生产经验证的引物集，用于这些试剂盒。我们还将优化和验证反应混合物配方，以实现对其他方法遗漏的低丰度RNA转录物的高效采样。还将开发用于上游选择定制引物组或基因组以及用于下游分析测序数据的分析工具。

DESCRIPTION (provided by applicant): We propose to develop and commercialize high sensitivity and accuracy RNA sequencing kits suitable for gene expression measurements in limiting input samples, such as single cells. During Phase I, we proposed the concept of using molecular barcodes in the form of DNA sequence tags to label individual molecules (or copies) of RNA for gene expression analysis in single cells. The Phase I project proved to be highly productive, and feasibility was demonstrated. Counting the barcodes provides an absolute, digital quantitative measure of the number of transcripts expressed. In addition, the barcodes are shown to be useful in correcting for PCR bias, a common challenge in small samples requiring DNA amplification protocols. We also determined a low RNA representation efficiency of 0.1-3.8% overall yield in current single cell RNA sequencing sample preparation methods, and demonstrate that with our cDNA amplification and barcoding approach, more accurate measurements are obtained at significantly higher yields of ~22.5%. These remarkable results indicate that our method can significantly improve on current techniques which suffer from large losses and have inaccuracies introduced by amplification distortions. Because measurement of gene expression levels is important in life science research and also in many clinical settings, our proposed product if successful, would contribute greatly to advancements in these areas. For Phase II, we propose to extend the validated concept into a product development program to produce high efficiency, user-friendly single-cell RNA sequencing kits. Specifically, we will perform assay development to produce validated sets of primers for inclusion in these kits. We will also optimize and validate reaction mix formulations to enable the high efficiency sampling of low abundance RNA transcripts that are missed by other methods. Analysis tools for the upstream selection of custom primer sets or gene panels, and for the downstream analysis of sequencing data will also be developed.