Transcriptional profiling and annotation of the Chlamydomonas genome

描述(申请人提供)：莱茵衣藻是光合作用和纤毛生物学基础发现的模式生物。它的基因组是联合基因组研究所的五个旗舰基因组之一，表明他们致力于完成和投资于维护数据库的基础设施。我们将通过使用在LLumina的平台上通过RNA-SEQ方法产生的转录组数据来增加JGI工作的价值，以a)获得更好的基因模型，b)在各种实验条件下识别差异表达的基因和替代转录物形式，以及c)推断基因组中每个基因的表达模式和每个转录物的绝对丰度。虽然已经对30万个EST进行了测序，但在16000个基因模型中，只有59%得到了EST覆盖的支持，只有21%同时具有5‘和3’非编码区信息。因此，许多基因模型，无论是计算预测的，还是基于同源性的，都是不正确的，或者充其量是不完整的。使用RNA-SEQ方法，我们获得了更大的转录组覆盖率，这可以显著提高注释的质量。在任何给定的条件下，转录丰度都有四个数量级的范围，但通过增加运行次数，我们甚至可以采样低丰度的转录本。因此，为了增加转录组的覆盖率，将收集在不同环境和生理条件下生长的细胞的转录序列数据，包括宏观和微观营养、pH、温度、光照强度、CO_2和02分压的变化，以及在不同发育情况下(配子发生、受精卵、细胞壁和鞭毛再生)。每个样本将被单独处理，以便我们可以比较每个样本内部和之间的特定RNA丰度，这将阐明与特定环境和发育条件相关的转录网络。初级序列数据将定期与相关元数据一起存放在NCBI SRA，导出的数据将加载到加州大学洛杉矶分校维护的公众可访问的浏览器上，并定期上载到Chlamybase和JGI基因组浏览器，以供长期公众访问。

DESCRIPTION (provided by applicant): Chlamydomonas reinhardtii is a model organism for fundamental discovery in photosynthesis and ciliary biology. Its genome is one of five flagship genomes at the Joint Genome Institute, indicative of their commitment to finishing and investment in infrastructure for maintaining the database. We will add value to the JGI effort by using transcriptome data, generated via RNA-seq methodology on lllumina's platform, to a) derive better gene models, b) identify differentially expressed genes and alternate transcript forms under various experimental conditions, and c) deduce the pattern of expression of every gene in the genome and the absolute abundance of every transcript. Although > 300,000 ESTs have been sequenced, only 59% of the ~16,000 gene models are supported by EST coverage, and only 21% have both 5' and 3' UTR information. Therefore, many gene models, both computationally predicted as well as homology-based are incorrect or at best incomplete. With RNA-seq approaches we obtain much greater coverage of the transcriptome, which can significantly elevate the quality of annotation. In any given condition, there is a range of four orders of magnitude for transcript abundance, but by increasing the number of runs we can sample even low abundance transcripts. Therefore, to increase the coverage of the transcriptome, transcript sequence data will be collected from cells grown under various environmental and physiological conditions, including variation in macro- and micro-nutrients, pH, temperature, light intensity, C02 and 02 partial pressures, and under different developmental situations (gametogenesis, zygote, cell wall and flagella regeneration). Each sample will be handled separately so that we can compare specific RNA abundance within and among each sample, which will elucidate transcription networks associated with specific environmental and developmental conditions. The primary sequence data will be deposited with associated metadata periodically at NCBI SRA and the derived data will be loaded onto a publicly accessible browser maintained at UCLA and periodically uploaded to Chlamybase and the JGI genome browser for long term public access.