TempO-Seq: A Multiplexed Gene Expression Profiling Platform

项目总结 该计划的第一阶段达到或超过成功标准，制定和实施 捕获自由Tempo-Seq分析，不仅能够测量mRNA，而且能够测量任何核酸靶标，验证 该平台具有完整的人类转录组分析和人类代理转录组分析，两者都 它们是作为商业产品推出的。代用分析是来自于 一种转录组，能够识别每一条已知的途径、化合物签名等，而不需要 来测量整个转录组。这些检测的性能超过了成功标准， 最初的计划是对多达1,000个信使核糖核酸进行分析，包括整个转录组分析 以30,247个RefSeq ID为目标(Affymetrix微阵列仅以30,654为目标)，以及替代测量 2733个基因。这一第二阶段属于NHGRI特别感兴趣的领域主题C：“基因组学工具 从新仪器到复杂的分子生物学试剂盒“。它将延长验证和交叉- 人体检测的平台研究，通过建立小鼠整体模型将检测扩展到小鼠 转录组Tempo-Seq分析，验证人类和小鼠替代物分析的准确性 通过测试不同的样本来识别途径并预测整个转录组中的基因变化 Hu和Mu Tempo-Seq全转录组分析，建立和验证重点基因集 从调查人员可以原样使用或有选择地组合的整个转录档案派生而来 一起或与代理进入为他们的研究定制的单一检测，实现允许的试剂盒 对离子激流进行测序，并全面实施Tempo-SeqR分析包，调查人员可以 用来处理他们的数据并导出文件，从中导出剂量和时间历程图，识别 差异表达基因和假阳性率，识别表达途径，并预测(从 哪些基因在整个转录组中没有被直接测量到 差异表达，所有这些都不需要生物信息学专家。这对调查人员的意义在于 Tempo-Seq分析可以提供相同的覆盖率和良好或更好的灵敏度来测量稀有 转录本为rna-seq，但可以以相同的成本对10到100个以上的样本进行测序，这是 重要的是，可以运行更多的样本来提供更高质量的数据和更详细的见解，或者 分析更多化合物、更多时间点或更多患者样本。调查人员可以让他们的 预算走得更远，获得更快的周转时间，例如在 MiniSeq，一种只需4.9万美元的自动测序仪，否则它无法运行整个转录组 RNA-seq分析，并获得更高质量的数据，而不需要RNA-seq所需的生物信息学专业知识。

PROJECT SUMMARY The Phase I of this program met or exceeded the success criteria, developing and implementing the capture free TempO-Seq assay, capable of measuring not just mRNA, but any nucleic acid target, verifying this platform with a whole human transcriptome assay and human surrogate transcriptome assay, both of which were launched as commercial products. The surrogate assay is a subset of genes from the whole transcriptome that is capable of identifying every known pathway, compound signatures, etc. without having to measure the whole transcriptome. The performance of these assays exceeded the success criteria and the original plans to implement assays of up to 1,000 mRNA targets, with the whole transcriptome assay targeting 30,247 RefSeq IDs (Affymetrix microarrays target only 30,654), and the surrogate measuring 2,733 genes. This Phase II falls under the NHGRI area of special interest Topic C: “Genomics tools ranging from new instruments to sophisticated molecular biology kits”. It will extend the validation and cross- platform studies for the human assays, extend the assay to mouse by establishing a mouse whole transcriptome TempO-Seq assay, validate the accuracy with which the human and mouse surrogate assays identify pathways and predict gene changes across the whole transcriptome by testing diverse samples in the Hu and Mu TempO-Seq whole transcriptome assays, establish and validate focused sets of genes derived from the whole transcriptome archives that investigators can use as-is or selectively combine together or with the surrogate into a single assay customized for their research, implement a kit permitting sequencing on the Ion Torrent, and fully implement a TempO-SeqR analysis package that investigators can use to process their data and derive a file from which to derive dose and time course graphs, identify differentially expressed genes and false positive rates, identify expressed pathways, and predict (from the surrogate) what genes across the whole transcriptome that are not directly measured are expected to be differentially expressed, all without requiring a bioinformatics expert. The significance to investigators is that the TempO-Seq assays can provide the same coverage and as good or better sensitivity to measure rare transcripts as RNA-seq but 10 to 100 more samples can be sequenced for the same cost, which is important because more samples can be run to provide higher quality data and more detailed insights, or to profile more compounds, or more time points, or more patient samples. Investigators can make their budgets go further, and get faster turn-around time such as running whole transcriptome assays on the MiniSeq, an automated sequencer costing only $49,000, which otherwise cannot run whole transcriptome RNA-seq assays, and get higher quality data, without the bioinformatics expertise required for RNA-seq.