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的RNA测序试剂盒，适用于有限输入样品（如单细胞）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单个RNA分子（或拷贝）的概念，以便在单细胞中进行基因表达分析。第一期项目被证明是高产的，并证明了可行性。计数条形码提供了表达的转录本数量的绝对数字定量测量。此外，条形码在纠正PCR偏差方面也很有用，这是需要DNA扩增方案的小样本中常见的挑战。我们还确定了当前单细胞RNA测序样品制备方法的低RNA表达效率，总产率为0.1-3.8%，并证明了我们的cDNA扩增和条形码方法，在高达~22.5%的产率下获得了更准确的测量结果。这些显著的结果表明，我们的方法可以显着改善目前的技术，这些技术遭受巨大的损失和放大失真带来的不准确性。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，我们提出的产品如果成功，将对这些领域的进步做出巨大贡献。对于第二阶段，我们建议将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。具体来说，我们将进行分析开发，以生产经过验证的引物，用于这些试剂盒。我们还将优化和验证反应混合配方，以实现其他方法无法实现的低丰度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面板的分析工具，以及用于测序数据的下游分析工具。

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.