Automated laser-induced fluorescence DNA sequencing: Equalizing signal-to-noise ratios significantly enhances overall performance

We have significantly optimized the performance of the Perkin-Elmer/Applied Biosystems (PE/ABI) 373Stretch DNA sequencer. Best results were obtained using 19% less ddNTPs and 75% more dNTPs relative to the manufacturer's recommended reaction mixture. Other changes included 4.25% (instead of 4%) acrylamide gels run at 38 W (instead of 40 W). These variations produced a significant equalization of the signal-to-noise profile, resulting in longer reads. The overall accuracy improvement for unedited pGEM 1000-base sequences analyzed by the ABI100 basecaller was 8.2%. High accuracy gains were observed in the 501- to 850-base region (6.0%) and, most significantly, from that point to the end of the sequence (41.6%). A decrease in accuracy was also found for the first 30 bases, which usually correspond to vector sequence. Our study has focused primarily on double-stranded DNA (dsDNA) using the DyeDeoxy Terminator chemistry kit and 48-cm well-to-read (WTR) gels since, in our hands, such a combination represents the quicker, most accurate, versatile, and productive choice for large-scale DNA sequencing. Nevertheless, our findings could be also exploited in other sequencing strategies using ssDNA templates, the DyePrimer chemistry, alternative: enzyme preparations, or different WTR lengths on the 373Stretch or other sequencing machines. (C) 1997 Academic Press.