Automatic Matrix Determination in Four Dye Fluorescence-based DNA Sequencing

The four dye fluorescence detection strategy is a widely used approach to automated DNA sequence analysis. An important aspect of data processing in this approach is the multicomponent analysis to deduce the concentrations of four fluorophores from fluorescence emission intensities at four different wavelengths. This requires knowledge of the correct transformation matrix M. The matrix M is a function both of the fluorophores employed and the fluorescence detection system. M is typically determined either by a calibration process with individual dyes, or by choosing four well-separated individual peaks corresponding to the four different dyes. Both are time-consuming and complicated procedures for routine use. An automatic scheme for finding M directly from raw sequence data is presented here. This facilitates data analysis and the underlying algorithm may also find utility in other multispectral applications.