Digital Gene Expression Analysis by 3’ End Sequencing

 描述(申请人提供)：深度测序的一个关键应用是RNA水平的基因表达分析，通常称为RNA-SEQ。RNA-seq的读数覆盖了mRNA序列的所有区域。然而，来自3‘末端区域的序列足以指示基因表达水平。所有的3‘末端测序方法都表明，大多数真核基因都表现出选择性的切割和多聚腺苷酸化(APA)，其中使用不同切割和多腺苷基化位点(PA)的RNA异构体可以从一个基因中产生。重要的是，不同的APA亚型具有不同的代谢，包括稳定性、定位和翻译。APA显然对基因表达分析提出了挑战。目前的RNA-SEQ方法无法捕捉到这一信息；其次，由于内部启动导致的错误PA识别导致了不准确的APA信息；第三，完整的基因表达分析需要考虑所有APA亚型。我们提出了一种新的方法，完全解决了错误启动、不完整的3‘端注释，并显著推动了迄今为止难以捉摸的单细胞RNA表达和Poly(A)尾长的研究。这项提议的最终目标是开发一种商业试剂盒，使非专业实验室能够以准确和敏感的方式检查3‘末端。

 DESCRIPTION (provided by applicant): A key application of deep sequencing is gene expression analysis at the RNA level, commonly known as RNA-seq. Reads from RNA-seq cover all regions of an mRNA sequence. However, sequences derived from the 3' end region are sufficient to indicate gene expression levels. All 3' end sequencing methods have revealed that most eukaryotic genes display alternative cleavage and polyadenylation (APA), where RNA isoforms using different cleavage and polyadenylation sites (pAs) can be generated from a gene. Importantly, different APA isoforms have been shown to have different metabolisms, including stability, localization and translation. APA clearly poses a challenge for gene expression analysis. Current RNA-seq methods cannot capture this information; second, false pA identification due to internal priming leads to inaccurate APA information; third, complete analysis of gene expression needs to take into consideration all APA isoforms. We have proposed a new method that completely addresses false priming, incomplete 3' end annotation and significantly propels research on single cell RNA expression and poly(A) tail length, which have so far been elusive. The final goal of this proposal is to develop a commercial kit, allowing non-specialized labs the ability to examine 3'ends in an accurate and sensitive manner.