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Genotyping at the HPCGG: Now and in the Future HPCGG Helix - February 2004 It is now well established that all complex diseases have a genetic basis. The initial goal of may research efforts in our community is the identification of genetic factors that cause susceptibility or resistance to these diseases. Scientists seeking to unravel the genetic contributions to complex human diseases like asthma, diabetes, and heart disease rely on a rapid influx of new genotyping technologies, identifying genes involved in common diseases in no small task. The most common approach is to look for associations between disease and specific genetic markers. Most assays look at single nucleotide polymorphisms (SNPs), the most common type of genetic variation. The pattern of SNPs for both alleles at a locus determines the genotype. Any two individuals vary at more than a million different SNPs scattered throughout the genome. A small fraction of this genetic variation is likely to explain the majority of differences between individuals, including their predisposition many common diseases. Sorting through this incredible amount of information requires high-throughout technologies. Many large-scale genotyping assays generate vast amounts of information and can incur significant expense. In its effort to increase the pace of discovery, the HPCGG constantly strives to incorporate new technologies for high throughput, low cost genotyping. Several new platforms are already in use at the Center, while others are in the pipeline. The HPCGG Genotyping Facility has relied heavily on the Sequenom® platform to detect genotypes by matrix-assisted laser desorption and ionization-time of flight (MALDI-TOF) mass spectrometry. Robotic automation and bar-coding speed the process, allowing multiplex PCR, clean-up, and a minisequencing reaction to be performed in a single well. Multiplexed reactions allow detection of as many as 5 SNPs per well of a 384 well plate. Automated software converts the mass spectrograph information to genotypes. After optimization of assay design and validation, Sequenom® provides a reliable, high throughput strategy for screening large numbers of samples for the same panel of genotypes. HPCGG-affliliated researchers Scott Weiss, M.D., M.S. and David Kwiatkowski, M.D., Ph.D. are pursuing a large-scale genotyping project to uncover genetic factors related to asthma. The project involves several investigators looking at sequence variations in over a dozen genes linked to asthma. Several hundred SNPs have been genotyped, mainly in multiplex format on the Sequenom® platform. Prior to the advent of high throughput genotyping at a reasonable cost, such studies were essentially inconceivable. The Center's Genotyping Facility also relies on Applied Biosystems' (ABI) Taqman® to identify target sites through fluorescence detection in real-time PCR. Single-plex SNP detection on the ABI 7900 allows allelic discrimination for multiple samples. Although not multiplexed, this method offers flexibility and reliability. New SNPs are being discovered all the time, and Taqman® is one approach that facilitates customization of assays to incorporate these newly discovered SNPs. The Center Also uses Pyrosequencing(TM) as another alternative for automated, high-throughput genotyping. After hybridization of a sequencing primer adjacent to the nucleotide of interest, deoxynucleotide triphosphates (dNTPs) are added individually. Incorporation of each individual dNTP causes the release of pyrophosphate, which can be detected as light through a chemical reaction involving a luciferase reporter. The readout is called a "pyrogram". Pyrosequencing(TM) offers a sensitive and reproducible method for SNP detection and genotyping. Dr. David Hunter of the HSPH and the BWH Channing Laboratory oversees the Taqman and Pyrosequencing methods. Applied Biosystems' new platform, SNPlex(TM), is geared for ultrahigh-throughput genotyping using the ABI 3730 and 3730xl DNA Analyzers. SNPlex(TM) uses an oligonucleotide ligation assay (OLA) to detect specific sequences, a universal PCR reaction to amplify ligated oligos, and a capillary electrophoresis step for genotyping. SNPlex(TM) increases throughput by multiplexing, and offers the convenience of operation on the same platform used for sequencing and linkage mapping. The Center plans to evaluate this technology in the very near future. The Affymetrix GeneChip® Human Mapping 10K Array chip is yet another genotyping platform in the pipeline. These resequencing chips allow an investigator to examine over 10,000 SNPs in a single assay. These 10K chips are currently being evaluated by Drs. Jon and Christine Seidman of HMS and BWH. Spacing these SNPs throughout the genome provides an economically feasible approach to lilnkage analysis or whole genome association studies. The next generation of Affymetrix chips may contain as many as 100,000 SNPs. All of these genotyping platforms provide quick turnaround of results in high-throughput formats. The balance between assay design flexibility, ease of data analysis, and cost will affect the choice of platform for a particular project. For more information about the Center's genotyping resources, visit our web site at www.hpcgg.org/genotyping. |