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Construction and sequencing of Expressed Sequence Tag (EST) libraries are commonly performed to reveal genes expressed in given tissues of an organism. The vast amount of sequencing data from the EST libraries requires effective analysis tools to translate such data into meaningful results for further research and development. While many EST analysis tools are available, most of them do not accommodate multi-project and multi-library environments and do not have enough tools to identify significant genes for the EST database. To address these issues, ESTplus, an integrated software tool collection, was developed to support better project management and provide more comprehensive collection of analysis tools. ESTplus allows its users to manage, create, modify, and remove EST libraries from their own projects. It consists of public programs necessary for performing a traditional EST analysis. In addition, the system was enhanced from other EST databases by including additional modules to help identifying functionally-significant or rare sequences as the following. First, library comparative analysis module finds the association between genes among EST libraries according to Gene Ontology (GO) terms using chi-square test (chi2 ). Second, integrative proteomics and EST data analysis module reversely identifies EST sequences that correspond to a short amino acid sequence resulted from a proteomic experiment. Third, a primer design module guides primer selection from gene contigs and/or sequences from the EST data. These enhanced features allow the better and easier utilization of the available EST data for functional genomics and translated biological research.