A genotyping system capable of simultaneously analyzing >1000 single nucleotide polymorphisms in a haploid genome

Abstract

A high-throughput genotyping system for scoring single nucleotide polymorphisms (SNPs) has been developed. With this system, >1000 SNPs can be analyzed in a single assay, with a sensitivity that allows the use of single haploid cells as starting material. In the multiplex polymorphic sequence amplification step, instead of attaching universal sequences to the amplicons, primers that are unlikely to have nonspecific and productive interactions are used. Genotypes of SNPs are then determined by using the widely accessible microarray technology and the simple single-base extension assay. Three SNP panels, each consisting of >1000 SNPs, were incorporated into this system. The system was used to analyze 24 human genomic DNA samples. With 5 ng of human genomic DNA, the average detection rate was 98.22% when single probes were used, and 96.71% could be detected by dual probes in different directions. When single sperm cells were used, 91.88% of the SNPs were detectable, which is comparable to the level that was reached when very few genetic markers were used. By using a dual-probe assay, the average genotyping accuracy was 99.96% for 5 ng of human genomic DNA and 99.95% for single sperm. This system may be used to significantly facilitate large-scale genetic analysis even if the amount of DNA template is very limited or even highly degraded as that obtained from paraffin-embedded cancer specimens, and to make many unpractical research projects highly realistic and affordable.

Figure 1.

Schematic illustration of the multiplex genotyping procedure. Only one SNP is shown. Primers and probes are shown as arrowed lines. Microarray spots are indicated as ellipsoids. (A) Amplification of the polymorphic sequence. Two allelic sequences use the same set of primers, P1 and P2. (B) Generation of ssDNA by using the primer-probes in both directions in separate tubes. Only the two allelic template strands in each reaction are shown. (C) ssDNA generated from B. (D) Addition of the ssDNA to the respective microarrays containing probes in different directions. (E) ssDNA templates hybridized to their probes on the microarrays. (F) Labeling probes by incorporating fluorescently labeled ddNTPs. (G) Labeled probes after washing off all other reagents.

Figure 2.

(A) A microarray image from genotyping one individual with Group II SNPs. Each probe was printed twice and shown as neighboring spots. Spots in red and green, homozygous; yellow, heterozygous; white, pink, and light green, spots with strong signal that have exceeded the linear range; and dark, low signal but not necessarily mean no signal or too low for genotype calls. (B) Scatter plot based on the color intensities from the microarray image shown in A. Two horizontal lines are the cutoffs (natural logarithms of the ratios [Cy3/Cy5] at 2 and -2) to divide the spots into three genotype groups. (C) A plot simply based on the two color intensities for the 24 samples (two spots for each sample) of an SNP. Values of the signal intensities indicated on the axes should be multiplied by 1000. Note that since different parameters are used, the color orientations are different in B and C.

Figure 3.

A microarray image from the analysis of single sperm with Group II SNPs. Each probe was printed twice as neighboring spots on the microarray. Spots in red and green, homozygous; yellow, heterozygous; white, pink, and light green, spots with strong signal that have exceeded the linear range; and dark, low signal but not necessarily mean no signal or too low for genotype calls. Yellow spots are either from SNPs that were not real because of the presences of a small portion of SNPs consisting of paralogous sequence variants in the databases (Cheung et al. 2003; Fredman et al. 2004), or from a low level (∼5%) of contamination as demonstrated in the previous studies (Cui et al. 1989; Goradia et al. 1991), which has been shown to be from oligonucleotides synthesized by the current hemi-open-oligonucleotide synthesis system. Note that heterozygous SNPs are treated as uninformative in genetic analyses with single sperm.