Stephan Pabinger1, Rene Snajder1, Robert Rader1, Heiko Eichhorn2, Zlatko Trajanoski1, Gerhard G. Thallinger1
1 Institute for Genomics and Bioinformatics, Graz University of Technology, Austria; 2 Development Anti-Infectives Microbiology, Sandoz GmbH, Kundl, Austria
We have developed QPCR, a web application for the analysis of data from qPCR experiments that integrates the complete analysis workflow including Cq and efficiency calculations, normalization, statistical analysis, and visualization. The application comprises parsers to automatically import data created by qPCR devices. Supported are files generated by the Applied Biosystems and Roche Lightcycler instruments software as well as a generic CSV file format. QPCR provides a graphical representation of the plate layout displaying sample, detector, and status information of each well on a grid. By selecting wells in the grid, charts of amplification and dissociation curves are displayed. Furthermore every property of a stored plate is fully editable allowing adjustment of data for later analysis. QPCR currently includes four algorithms for the calculation of the Cq value and six methods for the estimation of amplification efficiency values as described by Guescini et al. (2008), Ostermeier et al. (2003), Ramakers et al.(2003), Rutledge et al. (2004), Rutledge et al. (2008), Wilhelm et al. (2003), and Zhao et al. (2005). All algorithms and data file parsers used by the application are integrated through a plug-in mechanism, which allows simple extensions to support additional qPCR data formats and analysis methods. Further analysis steps include technical and biological replicate handling, incorporation of sample or target specific efficiency, normalization using single or multiple reference genes, inter-run calibration, and fold change calculation (see Hellemans et al. (2007)). In addition to the calculation of well-based efficiencies, efficiency can be determined based on serial dilution series using a graphical interface for the parameterization. QPCR includes standard and permutation based statistical tests to evaluate differences between samples groups and supports proper error propagation throughout all analysis steps. It provides a quality control method to evaluate applicability of the reference genes used for normalization (gene stability values and coefficients of variation). Results of all analysis steps are graphically displayed and can be exported as a data file to be used in external programs. The generated charts are highly customizable and are designed to be usable in publications without further manipulation. The web application includes an authentication and authorization system ensuring confidentiality and data security. Since 2007 the application has been deployed at several institutes and has been extensively tested and adapted to user requirements.
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