Cross-talk filtering in four dye fluorescence-based DNA sequencing.

Abstract

We have addressed two important issues of nonlinear cross-talk and baseline adjustment in DNA data processing. An important aspect in the processing of the four-dye fluorescence-based data is the cross-talk filtering. Typically, a matrix M, which is a function of the fluorophores and the fluorescence detection system, is used in the multicomponent analysis. In this deconvolution process the matrix is applied directly to the raw signal, on a linear cross-talk assumption. This necessitates the signal to be aligned to the baseline before the filter is applied. The various techniques used for aligning the raw data have the negative effect of adding distortion to the signal. An algorithm for cross-talk removal is presented in this paper. The algorithm uses the intensity difference of the signal rather than the actual value itself, thus making the cross-talk removal possible before the base line adjustment. In addition, a supplementary filtering step is proposed in order to account for the nonlinear nature of the cross-talk. This second step is based on a matrix T that accounts for the correlation of each of the signals with the other three. The overall result is a more precise presentation of the DNA data and less information loss through filtering.