Imaging as a tool for improving length and accuracy of sequence analysis in automated fluorescence‐based DNA sequencing

Abstract

A new method of signal analysis for automated fluorescence‐based DNA sequencing is presented. Signal resolution is a limiting factor in obtaining accurate sequence information beyond 400–450 nucleotides per gel lane. We have developed a computer program for the imaging of DNA bands in sequencing gels. The image analysis shows that distortions in the shapes of the bands decrease resolution of peaks observed in the standard data plots. Reconstruction of the undistorted band shape prior to signal analysis substantially improves the resolution of peaks and may improve the accuracy and length of the contiguous sequence read. Image analysis identified other factors limiting the accuracy and length of automated DNA sequence analysis and provided a tool for evaluating various remedies. Our techniques should also be applicable in other systems, for example, in gel electrophoresis of proteins and DNA restriction fragments, and in scranning densitometry.