RESUMO
We describe a web server for the accurate mapping of experimental tags in serial analysis of gene expression (SAGE). The core of the server relies on a database of genomic virtual tags built by a recently described method that attempts to reduce the amount of ambiguous assignments for those tags that are not unique in the genome. The method provides a complete annotation of potential virtual SAGE tags within a genome, along with an estimation of their confidence for experimental observation that ranks tags that present multiple matches in the genome. The output of the server consists of a table in HTML format that contains links to a graphic representation of the results and to some external servers and databases, facilitating the tasks of analysis of gene expression and gene discovery. Also, a table in tab delimited text format is produced, allowing the user to export the results into custom databases and software for further analysis. The current server version provides the most accurate and complete SAGE tag mapping source that is available for the yeast organism. In the near future, this server will also allow the accurate mapping of experimental SAGE-tags from other model organisms such as human, mouse, frog and fly. The server is freely available on the web at: http://dna.bio.puc.cl/SAGExplore.html.
Assuntos
Biologia Computacional/métodos , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sitios de Sequências Rotuladas , Software , Mapeamento Cromossômico , DNA Complementar/genética , Bases de Dados Genéticas , Etiquetas de Sequências Expressas , Internet , RNA Fúngico/genética , RNA Mensageiro/genética , RNA não Traduzido/genéticaRESUMO
BACKGROUND: In this study, we present a robust and reliable computational method for tag-to-gene assignment in serial analysis of gene expression (SAGE). The method relies on current genome information and annotation, incorporation of several new features, and key improvements over alternative methods, all of which are important to determine gene expression levels more accurately. The method provides a complete annotation of potential virtual SAGE tags within a genome, along with an estimation of their confidence for experimental observation that ranks tags that present multiple matches in the genome. RESULTS: We applied this method to the Saccharomyces cerevisiae genome, producing the most thorough and accurate annotation of potential virtual SAGE tags that is available today for this organism. The usefulness of this method is exemplified by the significant reduction of ambiguous cases in existing experimental SAGE data. In addition, we report new insights from the analysis of existing SAGE data. First, we found that experimental SAGE tags mapping onto introns, intron-exon boundaries, and non-coding RNA elements are observed in all available SAGE data. Second, a significant fraction of experimental SAGE tags was found to map onto genomic regions currently annotated as intergenic. Third, a significant number of existing experimental SAGE tags for yeast has been derived from truncated cDNAs, which are synthesized through oligo-d(T) priming to internal poly-(A) regions during reverse transcription. CONCLUSION: We conclude that an accurate and unambiguous tag mapping process is essential to increase the quality and the amount of information that can be extracted from SAGE experiments. This is supported by the results obtained here and also by the large impact that the erroneous interpretation of these data could have on downstream applications.
Assuntos
Mapeamento Cromossômico , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sitios de Sequências Rotuladas , Software , Biologia Computacional , DNA Complementar/genética , Bases de Dados Genéticas , Etiquetas de Sequências Expressas , RNA Fúngico/genética , RNA Mensageiro/genética , RNA não Traduzido/genética , Reprodutibilidade dos Testes , Mapeamento por Restrição , Análise de Sequência de DNA , Análise de Sequência de RNA , Transcrição GênicaRESUMO
OBJECTIVES: This study describes the diagnostic performance of a recently available multiplex PCR-based kit for the simultaneous detection and identification of Enterococcus faecium, Enterococcus faecalis, vanA, vanB, vanC1 and vanC2/C3 genes, directly from rectal swabs constituting the most complete existing molecular assay currently available. METHODS: The diagnostic performance of this assay was evaluated by a multicenter study involving three independent public hospitals and consisted in the analysis of 187 rectal swabs from patients at high risk for vancomycin-resistant enterococci colonization. RESULTS: When bacteria culture was used as the gold standard, the sensitivity, specificity, positive and negative predicted values for the assay were 96.8%, 76.0%, 67.7% and 97.9%, respectively. When a composite reference standard consisting of culture and DNA sequencing of PCR products was used as the gold standard, the sensitivity, specificity, positive and negative predicted values for the PCR-based assay were 97.8%, 96.9%, 96.7% and 97.9%, respectively. CONCLUSIONS: Based on these results, we conclude that this assay is considerably more sensitive than traditional microbiological methods for detecting vancomycin-resistant enterococci from rectal swabs. It is also much faster than culture. We believe that the implementation of this assay in routine clinical laboratories could help to reduce hospital-acquired vancomycin-resistant enterococci infections.