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

总结/摘要： 单细胞基因表达测定已经成为鉴定细胞功能亚型的重要工具， 以及由发育、疾病或治疗引起的特定亚型内的变化。然而，在这方面， 目前大多数方法昂贵、样品通量低并且依赖于寡聚-dT引发的逆转录。 此外，至今还没有一种方法能够使研究人员一致地测量低表达基因 从单细胞，这阻止了许多关键的生物标志物和重要的基因和分子的测量 途径。现有的单细胞测序方法的另一个问题是，许多细胞相关的DNA序列不稳定。 读段或者不能映射到转录物，或者映射到无信息的核糖体和线粒体基因。的 上述限制都导致大多数单细胞测序研究被限制在 相对少量的样品，并且阻止了它们在生物制药，临床， 翻译和应用市场。 在SBIR开发的第一阶段，我们实现了一种新的基于拆分池的组合工作流， 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。所有 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 在成千上万的细胞中的表达（和蛋白质）谱。TempO-LINC是一种无仪器方法 该系统每次运行可多路复用96个1，000个细胞样本，具有易于自动化的简单工作流程， 是所有商业单细胞平台中基因检测率最高的。至关重要的是，TempO-LINC减少了 每个样品的测定成本以及相关的测序成本;此外，尽管我们显示了测定 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 目的基因/途径-进一步显著降低测序成本。所有这些特征都定位于 TempO-LINC是一种潜在的颠覆性技术，研究人员，临床医生和药物开发人员可以使用它来 新的大规模应用/研究使用单细胞测序数千个样品，同时获得 提高数据质量。 我们在第二阶段提案中的目标是采用我们目前使用的基本TempO-LINC平台， 通过优化灵敏度、工作流程、多组学能力和 软件为了展示TempO-LINC的独特功能，我们将以10 X Chromium为基准进行测试 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化TempO-Seq测定/技术，第1阶段数据远远超过了所有 里程碑和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单元平台，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.