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.
Lario
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Instituto de Parasitología y Biomedicina, Consejo Superior de Investigaciones Científicas, Granada, Spain.
González
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Dorado
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G
D003854:Deoxyribonucleotides; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation / Q000379:methods / Q000706:statistics & numerical data; D005069:Evaluation Studies as Topic; D005453:Fluorescence; D007202:Indicators and Reagents; D007834:Lasers; D008838:Microcomputers; D017422:Sequence Analysis, DNA / Q000295:instrumentation* / Q000379:methods* / Q000706:statistics & numerical data
D003854:Deoxyribonucleotides; D007202:Indicators and Reagents