The SNPlex genotyping system: a flexible and scalable platform for SNP genotyping

Abstract

We developed the SNPlex Genotyping System to address the need for accurate genotyping data, high sample throughput, study design flexibility, and cost efficiency. The system uses oligonucleotide ligation/polymerase chain reaction and capillary electrophoresis to analyze bi-allelic single nucleotide polymorphism genotypes. It is well suited for single nucleotide polymorphism genotyping efforts in which throughput and cost efficiency are essential. The SNPlex Genotyping System offers a high degree of flexibility and scalability, allowing the selection of custom-defined sets of SNPs for medium- to high-throughput genotyping projects. It is therefore suitable for a broad range of study designs. In this article we describe the principle and applications of the SNPlex Genotyping System, as well as a set of single nucleotide polymorphism selection tools and validated assay resources that accelerate the assay design process. We developed the control pool, an oligonucleotide ligation probe set for training and quality-control purposes, which interrogates 48 SNPs simultaneously. We present performance data from this control pool obtained by testing genomic DNA samples from 44 individuals. in addition, we present data from a study that analyzed 521 SNPs in 92 individuals. Combined, both studies show the SNPlex Genotyping system to have a 99.32% overall call rate, 99.95% precision, and 99.84% concordance with genotypes analyzed by TaqMan probe-based assays. The SNPlex Genotyping System is an efficient and reliable tool for a broad range of genotyping applications, supported by applications for study design, data analysis, and data management.

FIGURE 1

A SNPlex Genotyping System assay protocol can be completed within two days. On the first day, the OLA reaction, exonuclease purification, and PCR amplification are performed. On the second day, the amplicons are immobilized on streptavidin-coated microtiter plates. ZipChute probes are hybridized to complementary ZipCode sequences, and non-hybridized ZipChute probes are washed away. The bound ZipChute probes are eluted and analyzed by CE.

FIGURE 2

GeneMapper software analyzes the electropherogram of a SNPlex system sample (A). Two neighboring peaks indicate the genotype of a particular SNP. The software creates for each SNP a genotype plot and uses a clustering algorithm to assign genotypes. SNP genotypes can be displayed as a Cartesian plot (middle ) in which the intensity of both peaks is measured on the x and y axis, or as a polar plot (right), in which the intensity of the peaks is measured on the x axis (length of arrow in Cartesian plot), and the ratio of both peak heights (angle of arrow in Cartesian plot) is measured on the y axis. The data point at the top of the arrow in the Cartesian plot is circled in the polar plot. B Cluster plots representing three SNPs from the control pool probe set, analyzing 44 genomic DNAs eight times on one 384-well plate. in each plot, 352 genotype calls are shown. The black square represents the position of a data point if no genomic DNA is used (non-template control).

FIGURE 3

User-defined SNPlex system assays are designed by means of a proprietary assay design pipeline, which can be accessed through the Applied Biosystems website. Ligation probes for individual SNP assays are designed using thermodynamic rules to ensure annealing specificity and efficiency. each probe undergoes extensive genome screening and is filtered for secondary structure or probe-probe chimeras. After the SNP-specific probes are designed, stringent rules are used to determine optimal probe-pool composition. assay annotations and allele calling information are provided as assay information files (AIF) on CD-ROM.