Biomedical subjects

Frank J Steemers

Publications and source records attributed to Frank J Steemers.

7 recordsLinked to original sources

PubMed2007

Whole genome genotyping technologies on the BeadArray platform.

The ability to simultaneously genotype hundreds of thousands of single-nucleotide polymorphisms (SNPs) in a single assay has recently become feasible due to innovative combinations of assay and array platform multiplexing. In this review, we describe the development of the Infinium whole genome genotyping technology and the BeadArray platform. We discuss the automated use and performance of a series of genotyping BeadChips, including data quality, technology scalability, and flexibility in designing array content. We describe high-density tag SNP-based Bead-Chips and various multi-sample BeadChip configurations with their respective applications. These technologies are enabling large-scale whole genome association studies that have the potential to revolutionize our ability to detect common genetic variants with a significant role in identifying disease-associated loci, proteins, biomarkers, and pharmacogenomic responses.

Biotechnology↗

PubMed2006

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

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 approximately 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.

Cell Line, Tumor↗

PubMed2006

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 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.

Animals↗

PubMed2006

Whole-genome genotyping with the single-base extension assay.

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.

DNA Primers↗

PubMed2006

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.

Automation↗

PubMed2005

A genome-wide scalable SNP genotyping assay using microarray technology.

Oligonucleotide probe arrays have enabled massively parallel analysis of gene expression levels from a single cDNA sample. Application of microarray technology to analyzing genomic DNA has been stymied by the sequence complexity of the entire human genome. A robust, single base-resolution direct genomic assay would extend the reach of microarray technology. We developed an array-based whole-genome genotyping assay that does not require PCR and enables effectively unlimited multiplexing. The assay achieves a high signal-to-noise ratio by combining specific hybridization of picomolar concentrations of whole genome-amplified DNA to arrayed probes with allele-specific primer extension and signal amplification. As proof of principle, we genotyped several hundred previously characterized SNPs. The conversion rate, call rate and accuracy were comparable to those of high-performance PCR-based genotyping assays.

Computational Biology↗

PubMed2005

Illumina, Inc.

Illumina, Inc., based in San Diego (CA, USA), is a genomics tool company that develops and markets integrated array-based systems and assays for a broad range of applications including genotyping, gene expression and epigenetics. Product offerings range from focused assay sets (up to 1,536 multiplexed assays) to whole-genome analysis (>100,000 assays/sample). Illumina's two microarray platforms, the Sentrix Array Matrix and the Sentrix BeadChip, are characterized by small (3 microm) feature size, dense feature packing (over 10 million features can be deployed on a single microarray) and the ability to analyze, in parallel, multiple samples on the same device. Illumina has developed a spectrum of proprietary assays (GoldenGate, Infinium, and DASL) for application on our microarray platform. These assays have been successfully employed for a number of applications, including collection of the majority of the Phase I genotyping data for the International HapMap Project. Illumina are focusing our activities on extending the applications of the above assays and developing new assays for future products. Illumina's goal is to deliver high-performance, high-throughput solutions that enable researchers to expand experimental scale while reducing the cost of large-scale research.

Gene Expression↗