Real-time PCR is a highly sensitive and powerful technology for the quantification of DNA and has become the method of choice in microbiology, bioengineering, and molecular biology. Currently, the analysis of real-time PCR data is hampered by only considering a single feature of the amplification profile to generate a standard curve. The current “gold standard” is the cycle-threshold (Ct) method which is known to provide poor quantification under inconsistent reaction efficiencies. Multiple single-feature methods have been developed to overcome the limitations of the Ct method; however, there is an unexplored area of combining multiple features in order to benefit from their joint information. Here, we propose a novel framework that combines existing standard curve methods into a multidimensional standard curve. This is achieved by considering multiple features together such that each amplification curve is viewed as a point in a multidimensional space. Contrary to only considering a single-feature, in the multidimensional space, data points do not fall exactly on the standard curve, which enables a similarity measure between amplification curves based ondistances between data points. We show that this framework expandsthe capabilities of standard curves in order to optimize quantificationperformance, provide a measure of how suitable an amplification curveis for a standard, and thus automatically detect outliers and increasethe reliability of quantification. Our aim is to provide an affordablesolution to enhance existing diagnostic settings through maximizingthe amount of information extracted from conventional instruments.
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