High-throughput genotyping with single nucleotide polymorphisms

Abstract

To make large-scale association studies a reality, automated high-throughput methods for genotyping with single-nucleotide polymorphisms (SNPs) are needed. We describe PCR conditions that permit the use of the TaqMan or 5' nuclease allelic discrimination assay for typing large numbers of individuals with any SNP and computational methods that allow genotypes to be assigned automatically. To demonstrate the utility of these methods, we typed >1600 individuals for a G-to-T transversion that results in a glutamate-to-aspartate substitution at position 298 in the endothelial nitric oxide synthase gene, and a G/C polymorphism (newly identified in our laboratory) in intron 8 of the 11-beta hydroxylase gene. The genotyping method is accurate-we estimate an error rate of fewer than 1 in 2000 genotypes, rapid-with five 96-well PCR machines, one fluorescent reader, and no automated pipetting, over one thousand genotypes can be generated by one person in one day, and flexible-a new SNP can be tested for association in less than one week. Indeed, large-scale genotyping has been accomplished for 23 other SNPs in 13 different genes using this method. In addition, we identified three "pseudo-SNPs" (WIAF1161, WIAF2566, and WIAF335) that are probably a result of duplication.

Figure 1

Genotyping results for the E298D SNP in the endothelial nitric oxide synthase gene. Data for 1699 genotypes are shown. (A) Raw data from the TaqMan PCR. (B) Data corrected for variation in pre-PCR fluorescence and k-means clustering. Fluorescence for the E allele is plotted along the X-axis (the dye is “FAM”) and for the D allele, labeled with the fluor “VIC”, along the Y-axis. “Rn” is fluorescence from the reporter dye divided by that from the passive reference dye. (Red squares) Samples homozygous for the E allele; (blue squares) samples homozygous for the D allele; (green squares) E/D heterozygotes; (black squares) “no DNA” controls or samples that failed to amplify. Arrows indicate samples with a very low conditional probability of belonging to that particular cluster.

Figure 2

Genotyping results for the CYP11B15 SNP. Data for 1699 genotypes are shown. (A) Raw data from the TaqMan PCR. (B) Output of the k-means clustering. Fluorescence values for the C and G alleles are plotted along the X and Y axes, respectively. (Red squares) C/C homozygotes; (green squares) G/C heterozygotes; (blue squares) G/G homozygotes; (black squares) “no DNA” controls or failed PCRs. Arrows indicate samples with a very low conditional probability of belonging to that particular cluster.

Figure 3

Pseudo-SNPs. (Left) Genomic DNA from 25–30 individuals was typed for the indicated SNPs. The axes are as in Figs. 1 and 2. Green dots are inferred to be heterozygotes; homozygotes are conspicuously absent. (Right) Synthetic templates bearing one (red dots) or the other allele (blue dots) were typed in duplicate using the same probes and primers used at left. Heterozygotes made by mixing equal amounts of homozygotes are shown in green.