Highly accurate small-RNA sequencing of single cells (RealSeq-SC)

单细胞高精度小 RNA 测序 (RealSeq-SC)

• 批准号: 10021698
• 负责人: Sergio Barberan-Soler
• 金额: $ 76.58万
• 依托单位: REALSEQ BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-05 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10021698
• 关键词: Applications Grants; Bar Codes; Benchmarking; Blood specimen; Breast; Breast Cancer cell line; Cancer Diagnostics; Cell Line; Cells; Chemicals; Cytolysis; Data; Detection; Development; Dropout; Event; Gene Expression; Gene Expression Regulation; Genome; Goals; Grant; Health; Individual; Lead; Libraries; Ligation; Manufacturer Name; Messenger RNA; Methods; MicroRNAs; Microfluidics; Modification; Neoplasm Circulating Cells; Noise; Oligonucleotides; Phase; Play; Population; Preparation; Protocols documentation; Publishing; RNA; Reaction; Reagent; Research Personnel; Role; Sampling; Signal Transduction; Small RNA; Sorting - Cell Movement; Technology; Tube; Whole Blood; base; circulating biomarkers; cost effective; design; dimer; experimental study; magnetic beads; miRNA expression profiling; neoplastic cell; next generation sequencing; novel; novel strategies; personalized therapeutic; phase 1 study; single cell analysis; single cell technology; single-cell RNA sequencing; therapeutic development; transcriptome; transcriptome sequencing; transcriptomics

Abstract The goal of this grant application is to develop the first commercially available library preparation kit for profiling small RNAs from single cells using NGS methods. Single-cell analyses of mRNA have allowed the identification of crucial differences between cells that were otherwise considered identical. These findings have shown that there is intrinsic “noise” in the regulation of gene expression within a population of cells that plays an important role in determining cell fates. Unfortunately, there is currently a lack of information about the cell-to-cell variability of levels of microRNAs that as gene expression regulators may also play a critical role. Indeed, there is no commercially available library preparation kit for miRNAs and other small RNAs that can profile single cells. We propose to quantify miRNAs from single cells using an advanced, proprietary “low input single adapter and circularization” technology that allows sensitive and unbiased detection. The core single adapter and circularization technology, for higher input quantities, demonstrated unbiased detection of over 70% of all miRNAs in a benchmark Universal miRNA pool, compared to ~35% from the best competitor kit. We have further developed this technology for single-cell analysis by creating a novel “low input version” that retains the detection accuracy even at single- cell levels. Data from our Phase I studies show that this “low input adapter” minimizes dropout events (a critical and common problem in single cell analysis) by increasing the efficiency of miRNA detection. Another major obstacle for single-cell miRNA sequencing is formation of adapter-dimers lacking miRNA inserts during library preparation that critically reduces the amount of useful miRNA sequencing reads. We employ three separate strategies to dramatically reduce the presence of adapter-dimers in the library. Also, our protocol performs all steps from cell lysis to final purification of amplified libraries in a single tube to reduce loss of miRNA from single-cells and to reduce the possibility of contamination of single-cell samples by environmental RNA. In Phase I we demonstrated proof-of-principle by detecting small RNAs from single-cells for three different cell lines. In Phase II, we will further develop and optimize our technology to significantly increase sensitivity and detection accuracy of miRNAs and other small RNAs from single cells for commercial viability. We will also develop a kit for single-cell small RNA-seq library preparation (RealSeq-SC).

摘要 这项拨款申请的目标是开发第一个商业化的图书馆制剂 使用NGS方法分析来自单细胞的小RNA的试剂盒。mRNA的单细胞分析 已经可以识别细胞之间的关键差异， 一模一样这些发现表明，在基因调控中存在内在的"噪音"， 在决定细胞命运中起重要作用的细胞群体内的表达。 不幸的是，目前缺乏关于细胞与细胞之间的差异性的信息， 作为基因表达调控因子的microRNA也可能发挥关键作用。其实，没有 用于miRNA和其他小RNA的市售文库制备试剂盒， 单细胞我们建议使用先进的、专有的"低水平"方法来定量来自单细胞的miRNA。 输入单适配器和循环"技术，允许灵敏和无偏检测。 核心的单适配器和循环化技术，为更高的输入量，展示了 在基准通用miRNA库中，无偏检测超过70%的所有miRNA， 从最佳竞争对手套件的约35%降低到约35%。我们进一步发展了这项技术， 通过创建一个新的"低输入版本"，即使在单一的分析， 细胞水平。从我们的第一阶段研究数据表明，这种"低输入适配器"最大限度地减少辍学 事件（单细胞分析中的关键和常见问题）， miRNA检测。单细胞miRNA测序的另一个主要障碍是 在文库制备期间缺乏miRNA插入物的衔接子二聚体， 有用的miRNA测序读数。我们采用三种不同的策略， 文库中衔接子二聚体的存在。此外，我们的方案执行从细胞裂解 在单管中最终纯化扩增文库以减少来自单细胞的miRNA损失 并降低单细胞样品被环境RNA污染的可能性。在 在第一阶段，我们通过检测来自单细胞的小RNA， 不同的细胞系。在第二阶段，我们将进一步开发和优化我们的技术， 增加来自单细胞miRNA和其它小RNA的灵敏度和检测准确性， 商业可行性。我们还将开发用于单细胞小RNA测序文库制备的试剂盒 （RealSeq-SC）。