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Nucleic Acids Research, Vol 25, Issue 14 2745-2751, Copyright © 1997 by Oxford University Press

ARTICLES

PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing

DA Nickerson, VO Tobe and SL Taylor
Department of Molecular Biotechnology, Box 357730, University of Washington, Seattle, WA 98195-7730, USA. debnick@u.washington.edu

Fluorescence-based sequencing is playing an increasingly important role in efforts to identify DNA polymorphisms and mutations of biological and medical interest. The application of this technology in generating the reference sequence of simple and complex genomes is also driving the development of new computer programs to automate base calling (Phred), sequence assembly (Phrap) and sequence assembly editing (Consed) in high throughput settings. In this report we describe a new computer program known as PolyPhred that automatically detects the presence of heterozygous single nucleotide substitutions by fluorescencebased sequencing of PCR products. Its operations are integrated with the use of the Phred, Phrap and Consed programs and together these tools generate a high throughput system for detecting DNA polymorphisms and mutations by large scale fluorescence-based resequencing. Analysis of sequences containing known DNA variants demonstrates that the accuracy of PolyPhred with single pass data is >99% when the sequences are generated with fluorescent dye-labeled primers and approximately 90% for those prepared with dye-labeled terminators.
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