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

摘要/摘要： 单细胞基因表达分析已经成为识别细胞功能亚型的关键工具， 以及由于发育、疾病或治疗治疗而导致的特定亚型内的变化。然而， 目前的大多数方法价格昂贵，样品吞吐量低，并且依赖于寡聚脱氧核糖核酸启动的逆转录。 此外，到目前为止，还没有一种方法能够使研究人员一致地测量低表达的基因 来自单个细胞，这阻碍了对许多关键生物标志物和重要基因和分子的测量 小路。现有单细胞测序方法的另一个问题是，许多与细胞相关的 阅读要么无法映射到转录本，要么映射到信息量不大的核糖体和线粒体基因。这个 上述限制都导致大多数单细胞测序研究被限制在 样本数量相对较少，并阻碍了它们在生物制药、临床和 翻译市场和应用市场。 在第一阶段的SBIR开发中，我们实现了一种新的基于组合拆分池的工作流， Tempo-LINC，它将细胞识别分子条形码添加到高灵敏度的基因表达探针上。全 同一细胞内的探针会收到相同的条形码，从而能够重建单细胞基因 数万个细胞的表达(和蛋白质)图谱。Tempo-LINC是一种无需乐器的方法 它每次运行多路传输96,000个细胞样本，具有易于自动化的简单工作流程，并具有 在所有商业单细胞平台中，基因检测率最高。关键是，Tempo-LINC降低了 每个样本的化验成本以及相关的测序成本；此外，尽管我们展示了化验 同时分析整个转录组(23,000个转录本)，它也可以仅针对测量 感兴趣的基因/途径--进一步大幅降低测序成本。所有这些功能都定位于 Tempo-LINC作为一种潜在的颠覆性技术，研究人员、临床医生和药物开发商可以使用 新的大规模应用/研究，使用跨越数千个样本的单细胞测序，同时获得 提高了数据质量。 我们在此第二阶段提案中的目标是利用我们目前的基本Tempo-LINC平台 通过优化敏感度、工作流程、多芯片功能和 软件。为了展示Tempo-LINC的独特功能，我们将以10倍Chromium为基准 使用PBMC的数据，并与我们的协作者/合作伙伴一起展示我们的技术的高影响力应用。 我们的方法依赖于现有的商业Tempo-Seq分析/技术，第一阶段数据远远超过所有 里程碑，以及坚实的知识产权基础。BioSpyder的Tempo-LINC构成了一个强大的 具有高技术成功潜力的单电池平台，将满足市场的关键需求。

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.