POLYPHRED - AUTOMATING THE DETECTION AND GENOTYPING OF SINGLE NUCLEOTIDE SUBSTITUTIONS USING FLUORESCENCE-BASED RESEQUENCING

Fluorescence-based sequencing is playing an increasingly important rol e in efforts to identify DNA polymorphisms and mutations of biological and medical interest, The application of this technology in generatin g the reference sequence of simple and complex genomes is also driving the development of new computer programs to automate base calling (Ph red), sequence assembly (Phrap) and sequence assembly editing (Consed) in high throughput settings, In this report we describe a new compute r program known as PolyPhred that automatically detects the presence o f heterozygous single nucleotide substitutions by fluorescence-based s equencing of PCR products, Its operations are integrated with the use of the Phred, Phrap and Consed programs and together these tools gener ate a high throughput system for detecting DNA polymorphisms and mutat ions by large scale fluorescence-based resequencing. Analysis of seque nces containing known DNA variants demonstrates that the accuracy of P olyPhred with single pass data is >99% when the sequences are generate d with fluorescent dye-labeled primers and similar to 90% for those pr epared with dye-labeled terminators.