title: Utility and accuracy of template-directed dye-terminator incorporation with fluorescence-polarization detection for genotyping single nucleotide polymorphisms.
abstract: There are little independent data available about how well single nucleotide polymorphism (SNP) genotyping technologies perform in the typical molecular genetics laboratory. We evaluated the utility and accuracy of a widely used technology, template-directed dye-terminator incorporation with fluorescence-polarization detection (FP-TDI), in a sample of 177 SNPs selected solely on the basis of map location. Genotypes were generated without optimization using standard protocols. Overall, 81% of the SNPs we studied generated readable genotypes by FP-TDI. Thirty-two SNPs were genotyped in duplicate by PCR-RFLP orfluorescent dye-terminator sequencing. Out of a total of 631 duplicate genotypes, no true discrepancies were detected. The true error rate has a 95% chance of lying between 0 and 6 out of 1000 genotypes. We also tested for deviations from Hardy-Weinberg Equilibrium in 33 SNPs genotyped in 50 unrelated individuals, and no significant deviations were detected. Our FP-TDI data were readily adaptable to automated genotype calling using our own method of cluster analysis, which assigns a probability score to each genotype call. We conclude that FP-TDI is both efficient and accurate. The method can easily fill the needs of SNP genotyping projects at the scale typically used for regional or candidate-gene association studies.
authors.affiliation: University of Chicago, IL, USA.
authors.forename: N
authors.initials: N
authors.lastname: Akula
authors.forename: Y S
authors.initials: YS
authors.lastname: Chen
authors.forename: K
authors.initials: K
authors.lastname: Hennessy
authors.forename: T G
authors.initials: TG
authors.lastname: Schulze
authors.forename: G
authors.initials: G
authors.lastname: Singh
authors.forename: F J
authors.initials: FJ
authors.lastname: McMahon
mesh_terms: D005454:Fluorescence Polarization; D005820:Genetic Testing / Q000379:methods* / Q000592:standards; D005838:Genotype; D006801:Humans; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D015203:Reproducibility of Results