A kit for single cell gene expression analysis

描述（由申请人提供）：基因表达的研究是我们理解细胞功能的基础。我们建议开发一种高度灵敏和精确的方法来测量单细胞中的全局基因表达。我们的新方法很简单，但能够准确地计算单个细胞内表达的所有基因的单个转录本。所获得的测量值是基因转录物的绝对数量，并且代表了相对于通常仅提供相对测量值的现代基因表达技术的显著改进。我们的方法是基于我们最近发表的“随机标记”的概念，我们通过用一组分子条形码随机标记样品中基因组DNA片段的每一个拷贝来验证这种新方法。标记后，用PCR扩增原始DNA片段，并通过大规模平行测序进行检测。计数不同条形码的数量揭示了该DNA片段的原始拷贝的数量，容易区分相同DNA序列的片段的多个拷贝与通过PCR复制产生的其自身的额外克隆。我们成功地将计数单个DNA分子的相同拷贝的挑战性任务转化为识别相同序列上存在的不同条形码数量的简单过程。我们目前的目标是第一阶段的技术发展成为单细胞基因表达分析的应用，定量测量是特别困难的，由于少量的起始材料存在。单细胞分析中的另一个重大挑战是所需的高度DNA扩增在基因丰度表示中产生偏差。我们通过在任何扩增步骤之前对分子进行条形码化来规避幅度失真的影响，并且对条形码而不是序列读数进行计数以确定存在的原始分子的数量。对于第二阶段，我们将开发一个商业检测试剂盒，其中包含试剂和协议，以进行该技术。我们公司由一支非常强大的成功创新者和科学家/工程师团队组成，在研究和产品商业化方面都取得了重大成就。成员包括单细胞/单分子研究领域的知名研究者，以及过去十年中最广泛使用的基因表达平台的关键发明者和开发者。此外，我们还与斯坦福大学基因组技术中心的科学家建立了合作关系，使我们能够使用这个世界一流研究机构提供的仪器和专业知识。

DESCRIPTION (provided by applicant): The study of gene expression is fundamental to our understanding of how cells function. We propose to develop a highly sensitive and precise method to measure global gene expression in single cells. Our novel method is simple, yet is capable of accurately counting individual transcripts across all genes expressed within a single cell. The measurements obtained are absolute numbers of gene transcripts, and represents a significant improvement over modern gene expression techniques which typically only provide relative measurements. Our method is based on the concept of "stochastic labeling" that we recently published where we validated the novel approach by randomly labeling every single copy of fragments of genomic DNA in a sample with a set of molecular barcodes. Once labeled, the original DNA fragments were amplified with PCR and detected by massively parallel sequencing. Counting the number of different barcodes reveals the number of original copies of that DNA fragment, easily distinguishing the plurality of copies of a fragment of identical DNA sequence from additional clones of itself created by PCR replication. We successfully transform the challenging task of counting identical copies of single DNA molecules into the simple process of identifying the number of different barcodes present on identical sequences. Our current objective for phase I is to develop the technique into an application for single cell gene expression analysis where quantitative measurements are especially difficult due to the small amount of starting material present. Another significant challenge in single cell analysis is that the high degree of DNA amplification required creates biases in the gene abundance representation. We circumvent the effects of amplitude distortions by barcoding molecules before any amplification steps, and counting barcodes instead of sequence reads to determine the number of original molecules present. For phase II, we will develop a commercial assay kit containing reagents and protocols to carry out the technique. Our company is comprised of an exceptionally strong team of successful innovators and scientists/engineers with significant achievements in both research and product commercialization settings. Members include an established investigator in the area of single cell/single molecule research, and a key inventor and developer of the most widely used gene expression platform over the past decade. Additionally, we have established collaborations with scientists at the Stanford University Genome Technology Center, giving us access to instruments and expertise available at this world class research facility.