Ebhardt H Alexander, Wiese Kay C, Unrau Peter J
Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, V5A 1S6 Burnaby, B.C., Canada.
BMC Bioinformatics. 2006 Apr 3;7:185. doi: 10.1186/1471-2105-7-185.
DNA sequencing is used ubiquitously: from deciphering genomes to determining the primary sequence of small RNAs (smRNAs). The cloning of smRNAs is currently the most conventional method to determine the actual sequence of these important regulators of gene expression. Typical smRNA cloning projects involve the sequencing of hundreds to thousands of smRNA clones that are delimited at their 5' and 3' ends by fixed sequence regions. These primers result from the biochemical protocol used to isolate and convert the smRNA into clonable PCR products. Recently we completed a smRNA cloning project involving tobacco plants, where analysis was required for approximately 700 smRNA sequences. Finding no easily accessible research tool to enter and analyze smRNA sequences we developed Ebbie to assist us with our study.
Ebbie is a semi-automated smRNA cloning data processing algorithm, which initially searches for any substring within a DNA sequencing text file, which is flanked by two constant strings. The substring, also termed smRNA or insert, is stored in a MySQL and BlastN database. These inserts are then compared using BlastN to locally installed databases allowing the rapid comparison of the insert to both the growing smRNA database and to other static sequence databases. Our laboratory used Ebbie to analyze scores of DNA sequencing data originating from an smRNA cloning project. Through its built-in instant analysis of all inserts using BlastN, we were able to quickly identify 33 groups of smRNAs from approximately 700 database entries. This clustering allowed the easy identification of novel and highly expressed clusters of smRNAs. Ebbie is available under GNU GPL and currently implemented on http://bioinformatics.org/ebbie/.
Ebbie was designed for medium sized smRNA cloning projects with about 1,000 database entries. Ebbie can be used for any type of sequence analysis where two constant primer regions flank a sequence of interest. The reliable storage of inserts, and their annotation in a MySQL database, BlastN comparison of new inserts to dynamic and static databases make it a powerful new tool in any laboratory using DNA sequencing. Ebbie also prevents manual mistakes during the excision process and speeds up annotation and data-entry. Once the server is installed locally, its access can be restricted to protect sensitive new DNA sequencing data. Ebbie was primarily designed for smRNA cloning projects, but can be applied to a variety of RNA and DNA cloning projects.
DNA测序应用广泛，从解读基因组到确定小RNA（smRNA）的一级序列。目前，克隆smRNA是确定这些重要基因表达调控因子实际序列的最常规方法。典型的smRNA克隆项目涉及对数百到数千个smRNA克隆进行测序，这些克隆在其5'和3'末端由固定序列区域界定。这些引物来自用于分离smRNA并将其转化为可克隆PCR产物的生化方案。最近我们完成了一个涉及烟草植物的smRNA克隆项目，需要对大约700个smRNA序列进行分析。由于没有易于使用的研究工具来输入和分析smRNA序列，我们开发了Ebbie来辅助我们的研究。
Ebbie是一种半自动的smRNA克隆数据处理算法，它首先在DNA测序文本文件中搜索任何两侧有两个恒定字符串的子串。该子串，也称为smRNA或插入片段，存储在MySQL和BlastN数据库中。然后使用BlastN将这些插入片段与本地安装的数据库进行比较，从而能够快速将插入片段与不断增长的smRNA数据库以及其他静态序列数据库进行比较。我们实验室使用Ebbie分析了来自一个smRNA克隆项目的大量DNA测序数据。通过其使用BlastN对所有插入片段进行的内置即时分析，我们能够从大约700个数据库条目中快速识别出33组smRNA。这种聚类使得能够轻松识别新的和高表达的smRNA簇。Ebbie遵循GNU GPL许可，目前可在http://bioinformatics.org/ebbie/上使用。
Ebbie是为具有约1000个数据库条目的中等规模smRNA克隆项目设计的。Ebbie可用于任何类型的序列分析，其中两个恒定引物区域位于感兴趣的序列两侧。插入片段的可靠存储及其在MySQL数据库中的注释，新插入片段与动态和静态数据库的BlastN比较，使其成为任何使用DNA测序的实验室中的强大新工具。Ebbie还可防止在切除过程中出现人工错误，并加快注释和数据输入速度。一旦在本地安装了服务器，可以限制对其的访问以保护敏感的新DNA测序数据。Ebbie主要是为smRNA克隆项目设计的，但也可应用于各种RNA和DNA克隆项目。
