Simple Two-Color Base-Calling Schemes for DNA Sequencing Based on Standard Four-Label Sanger Chemistry

Abstract

Two base-calling schemes for DNA sequencing are evaluated. Both are based on data collected from two broad-band emission channels derived from either one- or two-excitation channels. Standard four-dye Sanger reaction products are used in conjunction with capillary electrophoretic separation in a polymer matrix. Data acquisition is compatible with high light-throughput imaging and minimal data storage. In one scheme, commercial chromatographic software provides peak recognition and peak heights. The peak-height ratios from the two channels provide base-calling accuracies of 99.3% and 97.1% through 330 bp and 350 bp, respectively. In another scheme, ratiograms are derived from the two channels. The resulting step-like functions permit the calling of bases even when successive peaks are not resolved. The base-calling accuracy is 99% through 340 bp. Because of simplicity in implementation, either scheme should be readily applicable to high-speed, high-throughput DNA sequencing in capillary arrays.