[3] Illumina Universal Bead Arrays
Introduction
DNA polymorphisms and differences in gene expression provide the genetic basis for phenotypic variation. The systematic screening of an organism's genome for genetic variants and identification of environmental conditions that affect gene regulation and linking the former to a possible phenotype may advance our understanding of the underlying genetic or epigenetic mechanisms and lead to the identification of alleles (or haplotypes) and genes that contribute to phenotypic diversity. However, any comprehensive genome‐wide association studies would need to genotype a large number of single nucleotide polymorphisms (SNPs) (or measure expression of a large number of genes) in large sample sets (Botstein and Risch, 2003), thus requiring a system that combines very high throughput and accuracy with very low cost per SNP (or gene) analysis (Kwok 2003, Syvanen 2005). The technology described in this chapter provides a solution to these challenges by integrating a miniaturized array platform with a high level of assay multiplexing and scalable automation. The system uses the Sentrix BeadArray technology in combination with a solid‐phase, allele‐specific extension, followed by polymerase chain reaction (PCR) amplification to achieve high multiplex levels. The system is able to maintain high assay multiplex levels and high sample throughput in an informatically integrated production environment. Gunderson et al. (2005) described a genome‐wide scalable genotyping assay, the Infinium assay, which is also deployed on Sentrix arrays and allows simultaneous whole genome genotyping of over 100,000 SNP markers.
Section snippets
BeadArray Technology
Conventional microarrays are manufactured by spotting or synthesizing probes onto two‐dimensional substrates at known locations (Fodor 1991, Holloway 2002, Schena 1995). In contrast, BeadArray technology is based on the random self‐assembly of a bead pool onto a patterned substrate (Fan 2003, Michael 1998, Oliphant 2002). As a part of the array manufacturing process, a decoding process is implemented to map the precise location of a specific bead type on the array (Gunderson et al., 2004). The
Assay Flexibility
One key aspect of the GoldenGate genotyping assay design is incorporation of a universal IllumiCode sequence, allowing the assay products to be read out on a universal array (Chen 2000, Fan 2000, Gerry 1999, Iannone 2000). This approach offers substantial flexibility for assay content development. Any custom set of assays can be made on demand simply by building the complement of the IllumiCode into the SNP‐specific assay oligonucleotides (see Fig. 3, step 2). In addition, use of a universal
Conclusion
The GoldenGate genotyping platform combines a highly multiplexed genotyping assay with highly flexible Sentrix arrays to deliver high data quality and throughput. One of the most important features of the GoldenGate genotyping assay is that it genotypes directly on the genomic DNA and does not require prior PCR amplification of the genotyping target. It has been used in many genetic studies, including the International HapMap Project (The International HapMap Consortium, 2003), and has
Acknowledgments
We thank John Stuelpnagel, Nicky Espinosa, and Melanie Smith for critical reading of this chapter and constructive suggestions. We are indebted to the many other scientists and engineers at Illumina whose dedicated work has created the platforms and assays reviewed here.
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