Kenneth M. Kuhn

Whole‐Genome Genotyping

We have developed an array-based whole-genome genotyping (WGG) assay (Infinium) using our BeadChip platform that effectively enables unlimited multiplexing and unconstrained single nucleotide polymorphism (SNP) selection. A single tube whole-genome amplification reaction is used to amplify the genome, and loci of interest are captured by specific hybridization of amplified gDNA to 50-mer probe arrays. After target capture, SNPs are genotyped on the array by a primer extension reaction in the presence of hapten-labeled nucleotides. The resultant signal is amplified during staining and the array is read out on a high-resolution confocal scanner. We have employed our high-density BeadChips supporting up to 288,000 bead types to create an array that can query over 100,000 SNPs using the Infinium assay. In addition, we have developed an automated BeadChip processing platform using Tecans GenePaint slide processing system. Hybridization, washing, array-based primer extension, and staining are performed directly in Tecans capillary gap Te-Flow chambers. This automation process increases assay robustness and throughput greatly while enabling laboratory information management system control of sample tracking.

Whole-genome genotyping of haplotype tag single nucleotide polymorphisms

The International HapMap Consortium recently completed genotyping over 3.8 million single nucleotide polymorphisms (SNPs) in three major populations, and the results of studying patterns of linkage disequilibrium indicate that characterization of 300,000-500,000 tag SNPs is sufficient to provide good genomic coverage for linkage-disequilibrium-based association studies in many populations. These whole-genome association studies will be used to dissect the genetics of complex diseases and pharmacogenomic drug responses. As such, the development of a cost-effective genotyping platform that can assay hundred of thousands of SNPs across thousands of samples is essential. In this review, we describe the development of a whole-genome genotyping (WGG) assay that enables unconstrained SNP selection and effectively unlimited multiplexing from a single sample preparation. The development of WGG in concert with high-density BeadChips has enabled the creation of three different high-density SNP genotyping BeadChips: the Sentrix Human-1 Genotyping BeadChip containing over 109,000 exon-centric SNPs; the HumanHap300 BeadChip containing over 317,000 tag SNPs, and the HumanHap550 Beadchip containing over 550,000 tag SNPs.

Integrated Molecular Analyses of Biological Samples on a Bead-Based Microarray Platform

Molecular analyses of biological samples have traditionally been pursued in parallel, with those researchers studying genetic diversity having few technical approaches in common with those studying gene expression. Increasingly, scientists recognize the importance of integrating analytical technologies to further research, particularly into emerging fields such as epigenetics and the genetics of gene expression. In this chapter, we describe a suite of applications that take advantage of the Illumina® bead-based microarrays, all of which are read out on a single analytical instrument. The integration of whole genome genotyping, high throughput focused genotyping, whole transcriptome expression profiling, focused expression profiling of fresh or preserved tissues, allele-specific expression profiling and DNA methylation assays on the BeadArray™ Reader allows researchers to expand their perspectives, from whole genomes to single bases, from genetics to expression and on to epigenetics.