ADVANCE Atherosclerotic Disease Vascular
Function and Genetic Epidemiology

Phase II Genotyping Strategy
Initial Whole Genome Scan

In the first stage of the ADVANCE whole genome scan we will use the Infinium II Whole Genome Genotyping Assay that was developed by Illumina, Inc. (San Diego) to perform the genotyping. This platform is an ultra-highly multiplexed version of Illumina’s BeadArray technology 1-4 that allows hundreds of thousands of SNPs to be assayed in a single biochemical reaction. Here we describe the principles and practical steps of this method and how we will apply it to testing 550,000 SNPs spread throughout the genome for association with CAD.

Description of the Illumina technology

Illumina Technology

Illumina’s core technology, the BeadArray system, determines which of two bases is present at a polymorphic position in a complex sample, such as human genomic DNA. The ability of the assay to distinguish two different alleles is based on the extreme fidelity of DNA polymerase as it extends on a template in an allele-specific single-base extension reaction (See Figure). For each of the 550,000 SNPs, 50-base oligonucleotide probes are synthesized, immobilized on beads, and pools of these beads are arrayed on glass slides. Each of these oligos anneals to the 50 bases flanking the SNPs present in a genomic DNA sample. Following hybridization, a single base extension reaction is performed in parallel for all SNPs being interrogated on the BeadArray. The reaction uses dideoxynucleotides and two-color dye chemistry to distinguish the two alleles for a given SNP. Therefore, at a given bead type, which is designed to interrogate a particular SNP, a homozygous individual will generate a fluorescent signal for only one of the two colors (e.g. GG = all green or TT = all red), while a heterozygous individual will generate approximately equal fluorescent signals of both colors (e.g. TG = red and green). The Illumina BeadStation scanner is used to image the high-density arrays and the genotype calls are derived from the relative intensity of the fluorescent signals using BeadStudio software.

The strength of this technology lies in the fact that allelic discrimination is enzyme-based and not hybridization-based like other array technologies. High-fidelity DNA polymerases typically extend a primer with the correct base at the 3’ position with extremely high precision. This enables a degree of specificity and signal-to-noise ratio that greatly exceeds that of arrays based on perfect-match and mismatch oligo hybridization alone. The Illumina BeadArrays also benefit from the fact that an average of 30 beads of each type are present on every array, allowing for 30 independent measurements for each SNP, further increasing the likelihood of correct genotype calls.

The Infinium II assay itself has many features that make it attractive. First, it is remarkably sensitive; an amplification step combined with fluorescent labels plus a powerful scanner for detection, allow 550K genotypes to be performed with only about a microgram of genomic DNA. Second, the physical manipulations required to obtain the genotypes are extremely simple to perform, particularly with an automated liquid handling robot that we will use in the proposed work. Third, unlike several other ultra-high throughput genotyping methods, almost all regions of the human genome are amplified and assayable with the Illumina system, generating highly accurate genotypes with very low no call rates. In our hands, the Infinium II assay has performed remarkably well, with call rates >99.93% and accuracy rates >99.99%. To our knowledge, there is no other high-throughput genotyping technology that can match these performance measurements.

Audrey

Genotyping strategy for the second stage of the whole genome scan

We will perform more regional targeted follow up to this analysis by using a customizable version of the Illumina BeadArray technology to perform deeper genotyping for ~300 loci identified as most significantly associated with outcomes in the initial scan. Illumina’s custom Infinium assay uses the same principle as their fixed-content chips, but the arrays are produced on a smaller scale that allows custom (user-generated) SNPs to be assayed. The physical configuration and cost structures are such that ~ 12,000 SNPs are tested on each BeadArray at one quarter the price of the 550K chips. By using these arrays in the second phase of the study rather than the larger chips, we substantially reduce our genotyping costs.

SNPs for the 12K platform will be selected based on several criteria: strength of signal from the association study, size of potential regions and/or candidate genes involved, degree of linkage disequilibrium in these regions, presence of coding SNPs in these regions of reasonable frequency (say greater than 1%) found either in dbSNP or other databases or resources; also, the SNPs will comprise positive regions found from subtype-specific analyses. We believe the number 12,000 offers a good compromise between adequate coverage and costs.

References

  1. Shen R, Fan JB, Campbell D, et al. High-throughput SNP genotyping on universal bead arrays. Mutat Res 2005; 573:70-82.
  2. Murray SS, Oliphant A, Shen R, et al. A highly informative SNP linkage panel for human genetic studies. Nat Methods 2004; 1:113-7.
  3. Fan JB, Oliphant A, Shen R, et al. Highly parallel SNP genotyping. Cold Spring Harb Symp Quant Biol 2003; 68:69-78.
  4. Fan JB, Yeakley JM, Bibikova M, et al. A versatile assay for high-throughput gene expression profiling on universal array matrices. Genome Res 2004; 14:878-85.

 

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