An estimate of the crosstalk matrix in four-dye fluorescence-based DNA sequencing

Abstract

Color separation is an essential step of the data processing in the four-dye fluorescence detection strategy used in automated DNA sequencing. In this paper, we propose a model to describe the crosstalk phenomenon, and show how the assumptions of the model are supported by experimental data. The crosstalk matrix is estimated via a reparameterization based on a mapping between the distribution of fluorescence intensities and that of dye concentrations. An iterative algorithm is designed to implement the estimation. To evaluate the color-correction quality of a crosstalk matrix, we propose a quantitative measure based on the distribution of the color-corrected data. We illustrate this method by applying it to a sequencing trace of slab gel electrophoresis obtained at the Human Genome Center at Lawrence Berkeley National Laboratory, and that of capillary electrophoresis provided by the Department of Chemistry at UC, Berkeley. The accuracy of this method is also assessed by the bootstrap method.