Ebbie: automated analysis and storage of small RNA cloning data using a dynamic web serverReport as inadecuate




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BMC Bioinformatics

, 7:185

First Online: 03 April 2006Received: 06 December 2005Accepted: 03 April 2006

Abstract

BackgroundDNA sequencing is used ubiquitously: from deciphering genomes1 to determining the primary sequence of small RNAs smRNAs 2, 3, 4, 5. 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 ~700 smRNA sequences6. Finding no easily accessible research tool to enter and analyze smRNA sequences we developed Ebbie to assist us with our study.

ResultsEbbie 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 project6. Through its built-in instant analysis of all inserts using BlastN, we were able to quickly identify 33 groups of smRNAs from ~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-

ConclusionEbbie was designed for medium sized smRNA cloning projects with about 1,000 database entries 6, 7, 8.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, BlastN9 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 projects2, 3, 10, 11.

Abbreviations5-CP5- cloning primer

3-CP3- cloning primer

BLASTBasic Local Alignment Search Tool

GNU GPLGeneral Public License

NCBINational Center for Biotechnology Information

MySQLSQL: Standard Querying Language

mRNAmessenger RNA

ntnucleotide

PCRpolymerase chain reaction

rRNAribosomal RNA

tRNAtransfer RNA

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2105-7-185 contains supplementary material, which is available to authorized users.

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Author: H Alexander Ebhardt - Kay C Wiese - Peter J Unrau

Source: https://link.springer.com/



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