Automated Clone Evaluation for Functional Proteomics

功能蛋白质组学的自动克隆评估

• 批准号: 6819630
• 负责人: JOSHUA LABAER
• 金额: $ 35万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6819630
• 关键词: biomedical automation; complementary DNA; computer graphics /printing; computer human interaction; computer program /software; computer system design /evaluation; educational resource design /development; functional /structural genomics; gene expression; handbook; high throughput technology; interactive multimedia; molecular biology information system; molecular cloning; nucleic acid sequence; proteomics

DESCRIPTION (provided by applicant): Among the most compelling windfalls provided by the genome sequencing projects is the opportunity to begin to explore protein function space using systems approaches. Proteins play a critical role in biology and medicine; they are the targets of virtually all drugs. The study of protein function begins by expressing proteins from cloned copies of the corresponding cDNAs. Functional proteomics exploits new high-throughput technologies to study many proteins simultaneously using large collections of protein expression cDNA clones. However, the ability to interpret and rely upon the resulting data requires confidence that the clones accurately reflect the natural cDNA sequences. The successful automated production of these clone collections has exerted significant pressure to develop rapid and accurate methods for quality control. There is currently no software available that automates the sequence validation and evaluation of such clones, requiring that this be done by hand - a tedious and error prone process. The purpose of this proposal is to develop, maintain and openly distribute software that will automate and facilitate the process of biologically evaluating protein expression cDNA clones. This modular software will compare the assembled sequence of a cDNA clone to a user-specified reference sequence to identify and categorize all discrepancies. Most notably, the software will analyze: 1) the polypeptide effects of discrepancies between the clone and reference sequences, 2) if the discrepancies are due to natural polymorphisms, and 3) whether these differences render the clone unacceptable for a given experiment. Based upon user-defined penalties for each discrepancy type (truncation, conservative amino acid substitution, etc.), it will then recommend whether to pass, fail or require further review for a clone. By resetting the penalty scheme to meet different experimental requirements, users can re-evaluate the same clone set or any other sequenced clone set. This software will benefit molecular biologists who must validate the sequences of the cDNA clones or clone collections either produced in their labs or obtained from other sources. This software may also prove useful in identifying the natural genetic diversity of expressed mRNA. In this revised submission, we address the two major concerns expressed in the summary statement. First, we include letters of support from more than 20 labs currently building large clone collections who have expressed the need for this software. Second, we affirm that this software is exportable and not dependent on other software in our lab. Finally, we note that an alpha version of the software has now been implemented and tested on 6000 clones for yeast. In a sampling of 192 clones done by hand, the analysis was correct for all 192.

描述（由申请人提供）：基因组测序项目提供的最引人注目的意外收获之一是开始使用系统方法探索蛋白质功能空间的机会。蛋白质在生物学和医学中发挥着至关重要的作用；它们几乎是所有药物的目标。蛋白质功能的研究始于从相应 cDNA 的克隆拷贝表达蛋白质。功能蛋白质组学利用新的高通量技术，使用大量蛋白质表达 cDNA 克隆同时研究多种蛋白质。然而，解释和依赖结果数据的能力需要确信克隆准确地反映了天然 cDNA 序列。这些克隆集合的成功自动化生产给开发快速、准确的质量控制方法带来了巨大的压力。目前还没有可用的软件可以自动对此类克隆进行序列验证和评估，需要手动完成 - 这是一个繁琐且容易出错的过程。该提案的目的是开发、维护和公开分发软件，该软件将自动化并促进蛋白质表达 cDNA 克隆的生物学评估过程。该模块化软件将 cDNA 克隆的组装序列与用户指定的参考序列进行比较，以识别和分类所有差异。最值得注意的是，该软件将分析：1）克隆和参考序列之间差异的多肽效应，2）差异是否是由于天然多态性造成的，以及3）这些差异是否使克隆对于给定的实验不可接受。根据用户定义的对每种差异类型（截断、保守氨基酸取代等）的惩罚，它将建议是否通过、失败或需要对克隆进行进一步审查。通过重置惩罚方案以满足不同的实验要求，用户可以重新评估相同的克隆集或任何其他已测序的克隆集。该软件将使分子生物学家受益，他们必须验证实验室产生的或从其他来源获得的 cDNA 克隆或克隆集合的序列。该软件也可能有助于识别表达的 mRNA 的自然遗传多样性。在这份修订后的提交文件中，我们解决了摘要声明中表达的两个主要问题。首先，我们收录了来自 20 多个目前正在构建大型克隆集合的实验室的支持信，这些实验室都表达了对该软件的需求。其次，我们确认该软件是可导出的，并且不依赖于我们实验室的其他软件。最后，我们注意到该软件的 alpha 版本现已在 6000 个酵母克隆上实施并进行了测试。在手工完成的 192 个克隆样本中，所有 192 个克隆的分析都是正确的。