A framework for analysis of real-time nucleic acid amplification data using novel multidimensional standard curves

ABSTRACT Research into improving methods for absolute quantification of nucleic acids using standard curves has plateaued despite its positive, far-reaching impact on biomedical applications and clinical diagnostics. Currently, the mathematics involved in this mature area is restricted by the simplicity of conventional standard curves such as the gold standard cycle-threshold ( C t ) method. Here, we propose a novel framework that expands current methods into multidimensional space and opens the door for more complex mathematical techniques, signal processing and machine learning to be implemented. The heart of this work revolves around two new concepts: the multidimensional standard curve and its home - the feature space. This work has been validated using phage lambda DNA and standard qPCR instruments. We show that the capabilities of standard curves can be extended in order to simultaneously: enhance absolute quantification, detect outliers and provide insights into the intersection between molecular biology and amplification data. This work and its vision aims to maximise the information extracted from amplification data using current instruments without increasing the cost or complexity of existing diagnostic settings.