Showing papers by "Kevin L. Gunderson published in 2006"

High-resolution genomic profiling of chromosomal aberrations using Infinium whole-genome genotyping

Abstract: Array-CGH is a powerful tool for the detection of chromosomal aberrations. The introduction of high-density SNP genotyping technology to genomic profiling, termed SNP-CGH, represents a further advance, since simultaneous measurement of both signal intensity variations and changes in allelic composition makes it possible to detect both copy number changes and copy-neutral loss-of-heterozygosity (LOH) events. We demonstrate the utility of SNP-CGH with two Infinium whole-genome genotyping BeadChips, assaying 109,000 and 317,000 SNP loci, to detect chromosomal aberrations in samples bearing constitutional aberrations as well tumor samples at sub-100 kb effective resolution. Detected aberrations include homozygous deletions, hemizygous deletions, copy-neutral LOH, duplications, and amplifications. The statistical ability to detect common aberrations was modeled by analysis of an X chromosome titration model system, and sensitivity was modeled by titration of gDNA from a tumor cell with that of its paired normal cell line. Analysis was facilitated by using a genome browser that plots log ratios of normalized intensities and allelic ratios along the chromosomes. We developed two modes of SNP-CGH analysis, a single sample and a paired sample mode. The single sample mode computes log intensity ratios and allelic ratios by referencing to canonical genotype clusters generated from ∼120 reference samples, whereas the paired sample mode uses a paired normal reference sample from the same individual. Finally, the two analysis modes are compared and contrasted for their utility in analyzing different types of input gDNA: low input amounts, fragmented gDNA, and Phi29 whole-genome pre-amplified DNA.

Highly parallel genomic assays

Abstract: Recent developments in highly parallel genome-wide assays are transforming the study of human health and disease. High-resolution whole-genome association studies of complex diseases are finally being undertaken after much hypothesizing about their merit for finding disease loci. The availability of inexpensive high-density SNP-genotyping arrays has made this feasible. Cancer biology will also be transformed by high-resolution genomic and epigenomic analysis. In the future, most cancers might be staged by high-resolution molecular profiling rather than by gross cytological analysis. Here, we describe the key developments that enable highly parallel genomic assays.

Whole-genome genotyping with the single-base extension assay

Abstract: We describe an efficient, accurate and robust whole-genome genotyping (WGG) assay based on a two-color, single-base extension (SBE), single-nucleotide polymorphism (SNP)-scoring step. We report genotyping results for biallelic International HapMap quality control (QC) SNPs using a single probe per locus. We show scalability, throughput and accuracy of the system by resequencing homozygous loci from our 100k Human-1 Genotyping BeadChip.

Illumina universal bead arrays.

Abstract: This chapter describes an accurate, scalable, and flexible microarray technology. It includes a miniaturized array platform where each individual feature is quality controlled and a versatile assay that can be adapted for various genetic analyses, such as single nucleotide polymorphism genotyping, DNA methylation detection, and gene expression profiling. This chapter describes the concept of the BeadArray technology, two different Array of Arrays formats, the assay scheme and protocol, the performance of the system, and its use in large-scale genetic, epigenetic, and expression studies.

Whole-genome Genotyping

Abstract: 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 Tecan's GenePaint slide processing system. Hybridization, washing, array‐based primer extension, and staining are performed directly in Tecan's 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.

Abstract: 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. The goal of pharmacogenomics is to use individual’s genetic information to improve patient diagnosis, prognosis, and therapeutic intervention [1]. Genetic differences between individuals within a population underlie an individual’s predisposition to maintain health, develop disease, and respond efficaciously or adversely to drug therapy [2–4]. Human genetic variation is characterized, in part, by the presence of over 10 million relatively common, single nucleotide polymorphisms (SNPs) in the human genome. The linkage disequilibrium (LD) patterns observed in human population reveal a blocklike structure; SNPs within a high LD region (termed haploype block) tend to be transmitted together from generation to generation [5,6]. As such, a set of tag SNPs that serve as proxies for other SNPs can be chosen based on levels of correlation between their alleles [7,8]. The use of tag SNPs greatly reduces the genotyping burden in association studies, since a fewer number of tag SNPs need to be genotyped while maintaining the same information and power as if one had genotyped a much larger number of random SNPs [9]. The objective of the HapMap project was to delineate the haplotype block structure of the human genome and provide data for the identification of tag SNPs across multiple populations with ancestry from parts of Africa, Asia and Europe. The HapMap project completed both Phase I and II in late 2005 [10]; the resulting haplotype analysis of the data indicates that approximately 300,000 tag SNPs will provide good coverage for the European (CEU, Utah residents with ancestry from Northern and Western Europe) and Asian populations (CHB, Han Chinese in Beijing, China and JPT, Japanese in Tokyo, Japan), and a set of over 500,000 tag SNPs will provide improved coverage for European and Asian populations and good coverage for an African population (YRI, Yoruba in Ibadan, Nigeria) [10]. Infinium whole-genome genotyping platform We developed a whole-genome genotyping technology, known as the Infinium™ assay (Figure 1A), to enable genotyping of hundreds of thousand of SNPs on a single BeadChip™ substrate (slide). The whole-genome genotyping assay was developed to mirror the scalability of an array-based gene expression assay in which a simple singletube sample preparation allows readout of an entire transcriptome [11]. To make the assay inherently scalable, the Infinium assay employs a direct hybridization of a single-tube whole-genome amplified (WGA) DNA sample to an array of oligonucleotide capture probes (50-mers). The WGA step amplifies the input genomic DNA (gDNA) present at a few hundred nanograms by over 1000×, leading to hundreds of micrograms of WGA product. This high concentration of target drives the hybridization capture of the target loci to the probes on the array. Following capture of

Method for nucleic acid analysis

Abstract: PROBLEM TO BE SOLVED: To provide a method for monitoring expression of multiple screened genes. SOLUTION: The method for identifying differences of nucleic acid level between two or more samples comprises steps of: (a) providing one or more oligonucleotide arrays containing a probe oligonucleotide added on the surface; (b) hybridizing a nucleic acid sample with the above-mentioned one one more arrays to form a hybrid double bond between the nucleic acid in the nucleic acid sample and a probe oligonucleotide in the above-mentioned one or more array complementary to a subsequence thereof; (c) contacting the above-mentioned one or more arrays with a nucleic acid ligase; and (d) indicating the difference of nucleic acid levels with the differences of hybridization to determine the hybridization difference between nucleic acid samples. COPYRIGHT: (C)2006,JPO&NCIPI