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uAnalyze: Web-Based High-Resolution DNA Melting Analysis with Comparison to Thermodynamic Predictions
Nov.-Dec. 2012 (vol. 9 no. 6)
pp. 1805-1811
Z. L. Dwight, Dept. of Pathology, Univ. of Utah, Salt Lake City, UT, USA
R. Palais, Dept. of Math., Utah Valley Univ., Orem, UT, USA
C. T. Wittwer, Dept. of Pathology, Univ. of Utah, Salt Lake City, UT, USA
uAnalyzeSM is a web-based tool for analyzing high-resolution melting data of PCR products. PCR product sequence is input by the user and recursive nearest neighbor thermodynamic calculations used to predict a melting curve similar to uMELT (http://www.dna.utah.edu/umelt/umelt.html). Unprocessed melting data are input directly from LightScanner-96, LS32, or HR-1 data files or via a generic format for other instruments. A fluorescence discriminator identifies low intensity samples to prevent analysis of data that cannot be adequately normalized. Temperature regions that define fluorescence background are initialized by prediction and optionally adjusted by the user. Background is removed either as an exponential or by linear baseline extrapolation. The precision or, “curve spread,” of experimental melting curves is quantified as the average of the maximum helicity difference of all curve pairs. Melting curve accuracy is quantified as the area or “2D offset” between the average experimental and predicted melting curves. Optional temperature overlay (temperature shifting) is provided to focus on curve shape. Using 14 amplicons of CYBB, the mean +/ - standard deviation of the difference between experimental and predicted fluorescence at 50 percent helicity was -0.04 + / -0.48°C. uAnalyze requires Flash, is not browser specific and can be accessed at http://www.dna.utah.edu/uv/uanalyze.html.
Index Terms:
molecular biophysics,biology computing,data analysis,DNA,extrapolation,Internet,melting point,temperature shifting,uAnalyze,Web-based high-resolution DNA melting analysis,thermodynamic predictions,Web-based tool,high-resolution melting data analysis,PCR product sequence,recursive nearest neighbor thermodynamic calculations,melting curve prediction,LightScanner-96 data files,LS32 data files,HR-1 data files,fluorescence discriminator,temperature regions,linear baseline extrapolation,curve spread,maximum helicity difference,melting curve accuracy,optional temperature overlay,Computational biology,Bioinformatics,software,Melting curve analysis,high-resolution melting,biology and genetics,modeling and prediction
Citation:
Z. L. Dwight, R. Palais, C. T. Wittwer, "uAnalyze: Web-Based High-Resolution DNA Melting Analysis with Comparison to Thermodynamic Predictions," IEEE/ACM Transactions on Computational Biology and Bioinformatics, vol. 9, no. 6, pp. 1805-1811, Nov.-Dec. 2012, doi:10.1109/TCBB.2012.112
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