Signature Sequencing for Quantitative Expression Analysis and Gene Discovery

该项目的主要目标是展示一种名为“大规模平行特征测序”（MPSS）的新技术在植物基因表达量化方面的实用性。MPSS是一种快速产生17个碱基对序列标签的方法，这些标签精确地代表了给定组织中信使rna的群体。MPSS将对来自不同植物组织的大约8个文库进行测序，每个文库产生约50万个标签，总共有400万个标签。17-bp标签来自信使RNA或“转录本”的3'端，并为每个表达基因提供了几乎唯一的实验衍生标识符。给定基因文库中相同标签的数量精确地指示了该基因的表达水平。MPSS序列数据提供了整个“转录组”的定量或“数字”表达信息，避免了微阵列分析中固有的问题，如交叉杂交、探针序列的预选和低信号。定量表达数据的统计分析方法表明，这些数据是稳健的。与EST和SAGE技术获得的有限数据集相比，这些数据将是公共领域首次大规模的植物定量表达数据。当将这些标签与完全测序的基因组或大量est集合进行比较时，MPSS序列数据的信息量最大。为了充分利用MPSS技术，将从模式植物拟南芥（生态型哥伦比亚）中生成初始文库，并将MPSS标签与完整的基因组序列进行比较。这种比较确定了产生标签的单个基因。然后，这些数据将用于：量化和实验确认不同野生型和处理过的植物组织（包括茎、根、花序、角质层、花药、愈伤组织）的基因表达和mRNA转录；估计植物组织中选择性聚腺苷酸化的频率；研究共调控基因对，估计启动子强度和组织特异性；评估抗病反应的全球转录变化。通过该项目生成的序列标签将可通过网络访问，并且该接口将提供数据分析工具。这些数据也将被放入公共表达数据库（如国家生物技术信息中心的数据库，‘NCBI’），以便与微阵列数据进行比较，从而促进对这些文库中存在的任何单个基因或所有基因的表达分析。这些实验为在植物中进行大规模平行特征测序以进行基因表达的全球分析提供了概念证明。可交付成果本项目的可交付成果以电子方式提供：a href=http://www.dbi.udel.edu/mpss http://www.dbi.udel.edu/mpss /a

The primary goal of this project is to demonstrate the utility of a novel technology called 'massively parallel signature sequencing' (MPSS) for the quantification of gene expression in plants. MPSS is a rapid method to produce 17 base pair sequence tags that are precisely representative of the population of messenger RNAs in a given tissue. Approximately eight libraries from diverse plant tissues will be sequenced by MPSS, generating ~500,000 tags per library, for a total of four million tags. The 17-bp tag is derived from the 3' end of a messenger RNA or 'transcript' and provides a virtually unique, experimentally derived identifier for each expressed gene. The number of identical tags in a library for a given gene is precisely indicative of the level of expression of that gene. The MPSS sequence data provide quantitative or 'digital' expression information for the entire 'transcriptome', avoiding problems inherent in microarray analysis such as cross-hybridization, pre-selection of probe sequences and low signal. Statistical methods for the analysis of quantitative expression data have demonstrated that these data are robust. In comparison to the limited sets of data derived by the EST and SAGE techniques, these data will be the first large-scale quantitative expression data for plants in the public domain. The MPSS sequence data is most informative when the tags are compared to either a completely sequenced genome or to large collections of ESTs. To take full advantage of the MPSS technology, the initial libraries will be generated from the model plant Arabidopsis (ecotype Columbia) and the MPSS tags compared to the complete genomic sequence. This comparison identifies the individual genes from which the tags are derived. These data then will be used to: quantify and experimentally confirm gene expression and mRNA transcripts in diverse wildtype and treated plant tissues (including shoot, root, inflorescence, silique, anthers, callus); estimate the frequency of alternative polyadenylation in plant tissues; study co-regulated gene pairs and estimate promoter strength and tissue specificity; assess global transcriptional changes in the disease resistance response. Sequence tags generated through this project will be accessible via the web, and the interface will provide tools for data analyses. These data also will be placed in public expression databases (such as those at the National Center for Biotechnology Information, 'NCBI') for comparison to microarray data, facilitating the expression analysis of either any single gene or all genes present in these libraries. These experiments constitute a proof of concept for massively parallel signature sequencing in plants for the global analysis of gene expression.DeliverablesThe deliverables for this project are available electronically at:a href=http://www.dbi.udel.edu/mpss http://www.dbi.udel.edu/mpss /a