TempO-LINC high throughput high sensitivity single cell gene expression profiling assay Ph II

TempO-LINC 高通量高灵敏度单细胞基因表达谱分析第二阶段

• 批准号: 10699784
• 负责人: BRUCE E. SELIGMANN
• 金额: $ 124.13万
• 依托单位: BIOSPYDER TECHNOLOGIES, INC.
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-22 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10699784
• 关键词: Address; Adoption; Affect; Automation; Bar Codes; Benchmarking; Bioinformatics; Biological Assay; Biological Markers; Biological Sciences; Cell Nucleus; Cells; Chromium; Clinical; Communicable Diseases; Complex; Computer software; Data; Data Analyses; Detection; Development; Differentiated Gene; Disease; Dissociation; Foundations; Gene Expression; Gene Expression Profiling; Genes; Heterogeneity; Immunology; Intellectual Property; Malignant Neoplasms; Manuals; Maps; Marketing; Measurement; Measures; Methods; Mitochondria; Molecular; Pathway interactions; Patient-Focused Outcomes; Peripheral Blood Mononuclear Cell; Pharmaceutical Preparations; Phase; Phenotype; Population; Positioning Attribute; Proliferating; Proteins; Protocols documentation; Research; Research Personnel; Reverse Transcription; Ribosomes; Running; Sampling; Small Business Innovation Research Grant; Solid; Technology; Tissues; Transcript; Update; Validation; Variant; Vision; Work; combinatorial; commercial launch; commercialization; cost; data quality; graphical user interface; improved; instrument; interest; mRNA Expression; multiple omics; novel; oligo (dT); open source; prevent; protein expression; reconstruction; research study; sample fixation; sequencing platform; single cell analysis; single cell sequencing; statistics; success; tool; transcriptome; treatment response; user-friendly

Summary/Abstract: Single cell gene expression assays have become critical tools for identifying functional subtypes of cells, as well as changes within specific subtypes resulting from development, disease or therapy treatment. However, most current methods are expensive, low in sample throughput and rely on oligo-dT primed reverse transcription. Furthermore, there is no method to-date that enables investigators to consistently measure low expressed genes from single cells, which prevents measurements of many key biomarkers and important genes and molecular pathways. Another problem with existing single-cell sequencing methods is that many of the cell-associated reads either fail to map to transcripts or map to uninformative ribosomal and mitochondrial genes. The aforementioned limitations have all contributed to most single-cell sequencing studies being restricted to relatively small numbers of samples and have prevented their wider scale adoption in biopharma, clinical, translational and applied markets. In Phase 1 SBIR development, we implemented a novel combinatorial split-pool-based workflow, TempO-LINC, that adds cell-identifying molecular barcodes onto high-sensitivity gene expression probes. All probes within the same cell receive an identical barcode, enabling the reconstruction of single-cell gene expression (and protein) profiles across tens of thousands of cells. TempO-LINC is an instrument free approach that multiplexes 96 1,000 cell samples per run, has a simple workflow that is amenable to automation and has the highest gene detection rate of any commercial single-cell platform. Critically, TempO-LINC reduces both the per sample assay cost as well as associated sequencing costs; furthermore, although we show the assay simultaneously profiles the whole transcriptome (23,000 transcripts), it can also be targeted to measure only genes/pathways of interest – dramatically reducing sequencing costs yet further. All of these features position TempO-LINC as a potential disruptive technology that researchers, clinicians and drug developers can use for new large-scale applications/studies using single-cell sequencing across thousands of samples while obtaining improved data quality. Our objective in this Phase 2 proposal is to take the basic TempO-LINC platform that we are currently running and ready it for commercialization by optimizing the sensitivity, workflow, multi-omic capability and software. To demonstrate the unique capabilities of TempO-LINC, we will benchmark it against 10X Chromium data using PBMCs and demonstrate a high-impact application of our technology with our collaborators/partners. Our approach relies on existing commercial TempO-Seq assays/technology, Phase 1 data that far exceeded all milestones, and a solid foundation of intellectual property. BioSpyder’s TempO-LINC constitutes a powerful single-cell platform with high potential for technical success, which will fill a critical need in the marketplace.

摘要/摘要： 单细胞基因表达测定已成为鉴定细胞功能亚型的关键工具， 以及由发育、疾病或治疗引起的特定亚型的变化。 目前大多数方法价格昂贵、样品通量低并且依赖于oligo-dT 引发的逆转录。 此外，迄今为止还没有方法使研究人员能够一致地测量低表达基因 来自单细胞，这阻止了许多关键生物标志物以及重要基因和分子的测量 现有单细胞测序方法的另一个问题是许多细胞相关的途径。 读取要么无法映射到转录本，要么映射到无信息的核糖体和线粒体基因。 所讨论的局限性都导致大多数单细胞测序研究仅限于 样本数量相对较少，阻碍了其在生物制药、临床、 转化和应用市场。 在第一阶段 SBIR 开发中，我们实施了一种新颖的基于组合拆分池的工作流程， TempO-LINC，将细胞识别分子条形码添加到高灵敏度基因表达探针上。 同一细胞内的探针接收相同的条形码，从而能够重建单细胞基因 TempO-LINC 是一种无需仪器的方法。 每次运行可多重处理 96 1,000 个细胞样本，工作流程简单，适合自动化，并且具有 TempO-LINC 是所有商业单细胞平台中最高的基因检测率。 每个样本的分析成本以及相关的测序成本；此外，我们还展示了分析方法 同时分析整个转录组（23,000 个转录本），也可以有针对性地仅测量 感兴趣的基因/通路——进一步显着降低测序成本。 TempO-LINC 作为一项潜在的颠覆性技术，研究人员、新手和药物开发人员可以将其用于 使用单细胞测序对数千个样本进行新的大规模应用/研究，同时获得 提高数据质量。 我们第二阶段提案的目标是采用我们目前的基本 TempO-LINC 平台 通过优化灵敏度、工作流程、多组学能力和 为了展示 TempO-LINC 的独特功能，我们将其与 10X Chromium 进行基准测试。 使用 PBMC 收集数据，并与我们的合作者/合作伙伴一起展示我们技术的高影响力应用。 我们的方法依赖于现有的商业 TempO-Seq 检测/技术，第一阶段数据远远超过了所有 BioSpyder 的 TempO-LINC 的里程碑和坚实的知识产权基础构成了强大的力量。 具有很高技术成功潜力的单细胞平台，将满足市场的关键需求。