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Association Study Application Note nn nn Solutions for Each Step of an Association Study WHOLE-GENOME SCAN Overview Scan whole-genome hypothesis free Affymetrix offers solutions for each step of a typical whole-genome association study—from initial discovery, to replication and fine mapping, sequence analysis, and functional testing of genes. Researchers worldwide are now using these tools to identify the genetic causes of a wide range of common diseases such as autism, obesity, cancer, asthma, and diabetes. Products: • GeneChip® Mapping 500K Array Set FINE MAPPING/ REPLICATION WHOLE-GENOME SCAN Products: • Custom Targeted Genotyping In this initial step, an unbiased subset of all SNPs are genotyped on a microarray to determine which are statistically associated with a given phenotype— a predisposition to disease, drug response, diagnosis, or prognostic outcome. SEQUENCE ANALYSIS FINE MAPPING/REPLICATION Reduce to candidate region/ genes; repeat in another population Identify candidate nucleotide variants Products: • GeneChip CustomSeq® Arrays FUNCTIONAL TESTS Understand the function Products: • Gene Expression • Exon Expression • Gene Regulation • Full Transcriptome In this second step, additional SNPs in the regions identified during the initial whole genome scan are analyzed for statistical association with the phenotype. SNPs are re-genotyped in additional sample populations, or in the same population, to replicate the initial discoveries. SEQUENCE ANALYSIS In this third step, genomic regions are chosen for detailed sequence analysis to identify all SNPs in these regions within a particular population. Regions are chosen based on proximity to statistically significant SNPs and, often, functional annotations. Those SNPs can then be analyzed for statistical association with the phenotype and researchers can ultimately pinpoint one or more causal variants. FUNCTIONAL TESTING In this final step, researchers look to verify and explore the association between the causal variant that has been discovered and the phenotypic effects. LOWER COSTS. MORE SAMPLES. HIGHER GENETIC POWER. The number of samples genotyped in your study is the single most important factor in powering your whole-genome scan. The GeneChip® Human Mapping 500K Array Set reduces genotyping costs to $250 per sample,* which allows you to dramatically increase the number of samples you can run in a single study. More samples translate into higher genetic power to detect true associations (Figures 1 and 2). U.S. list price * 1,000,000 SNPS IN 2007 Use a platform that will grow with your future needs. Affymetrix will introduce a 1 million-SNP product by the first quarter of 2007. Additionally, by the end of 2006, Affymetrix will begin offering a singlearray genotyping product for wholegenome association studies to replace the Mapping 500K Array Set. This new product, developed in collaboration with the Broad Institute of MIT and Harvard, will allow researchers to increase throughput, devote more resources toward performing larger experiments, and analyze more copy number variation in their studies. CONTROLS DATABASE Access 6 billion control genotypes to supplement your data—without spending a penny more. Over 10 investigators worldwide have agreed to make their 500K genotyping data—more than 13,000 samples from multiple ethnic backgrounds— accessible to other scientists. Watch an Affymetrix UserForum interview with Stefan Schreiber, Christian-Albrechts-University, lead investigator and contributor to the database, at www.affymetrix. com/userForum/news/collaborations/schreiber.uf Analysis has shown that doubling the number of control genotypes can result in a 20 percent increase in genetic power (Figure 3). Also, by accessing these freely nn nn 2 GENOTYPING Figure 1: This chart illustrates the influence of sample size on genetic power for varying percentages of genome coverage. Mapping 500K Mapping 500K Array Set Array Set 2X Samples 1X Samples 550K Tag Product 1X Samples 100% COPY NUMBER AND SNP VARIATION Analyze SNP and copy number variation in a single assay. A growing number of geneticists are studying germ line copy number variation in their association or linkage studies. The Mapping 500K Array Set allows you to identify disease-causing copy number variation on the same arrays, with no additional cost. Genotype information, in addition to quantitative copy number information, allows researchers to distinguish between copy-neutral events, such as Uniparental Disomy (UPD). 90% 80% 70% Genome Coverage Study Design and Genetic Power available data, you may be able to run fewer controls. Researchers using the controls database will be able to select age- and sex-matched controls from ethnic backgrounds similar to their cases, and add them to their current study. 60% 50% 40% 30% 20% 10% 0% Using three marker analysis Listen to an Affymetrix Microarray Bulletin symposium with Stanley Nelson, UCLA, on disease-associated copy number variation at www.microarraybulletin.com/ community/article.php?p=134 More resolution provides more power to detect chromosomal aberrations and better define boundaries and break points. The Mapping 500K Array Set provides a mean resolution of 5.8kb and Better power than r2=0.8 Equivalent power r2=0.8 Worse power than r2=0.8 a median marker distance of 2.5kb, giving scientists the highest physical coverage of the genome for their study. Affymetrix offers a growing family of software solutions for copy number analysis, including standardized thirdparty software providers: Figure 2: This example power calculation illustrates how more samples impact genetic power. Power: Multiplicative Genetic Relative Risk 1.3 1 0.9 Power to detect association at 0.05 Whole-Genome Scan 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 200 300 400 $180k study at $600 per sample = 300 samples 500 600 700 800 900 1000 1100 1200 1300 1400 1500 $180k study at $250 per sample = 720 samples Number of cases (for 1:1 case:control study) · The Affymetrix ® Copy Number Analysis Tool (CNAT) and the Affymetrix Integrated Genome Browser (IGB) are available for download at www.affymetrix.com. CNAT supports copy number and Loss of Heterozygosity (LOH) analysis of Mapping Array data, including 10K, 50K, and 100K. Look for expanded support for the 500K in late 2006. · Partek® Genomics Solution™ is the first commercial software application available for copy number analysis in support of Mapping 10K, 100K, and 500K arrays. · CNAG, from Dr. Seishi Ogawa at the University of Tokyo, and dChip SNP, from Dr. Cheng Li at Harvard University and the Dana Farber Cancer Institute, are both academic software tools available for copy number and LOH analysis of Affymetrix Mapping Arrays, including 10K, 100K, and 500K. Genotyping Software, Downstream Analysis, and Sample Tracking Affymetrix uses an open informatics platform and partners with a community of over 25 software development partners, to provide flexible and innovative genotyping software solutions for researchers in different laboratory environments, with different software development resources. GENECHIP® OPERATING SYSTEM (GCOS) Affymetrix GeneChip® Operating Software (GCOS) uses a database structure for storage and management of samples, sample attributes, associated arrays and the resulting data. GCOS is an out-ofthe-box software solution for primary data generation. Using a standard Microsoft Windows XP workstation, GCOS can associate sample attributes with the resulting files. These data are stored in a lightweight, freely available database. INPUT DATA AND SAMPLE ATTRIBUTES QUICKLY AND EASILY INTO GCOS Today, Affymetrix supports a number of methods to push data into GCOS through a variety of input data types—from simple CSV or XML files, to ties to external databases through embedded libraries. Once the data reside within GCOS, sample attributes are natively associated with the resulting files, and samples run on multiple arrays are properly associated. Researchers can use GCOS as the master data repository, or use existing methods to pull data out of GCOS into another data repository. To learn more about integration Figure 3: Doubling the number of controls can result in a 20 percent increase in genetic power. options, please contact the Affymetrix Developers’ Network Mailbox: devnet@ affymetrix.com. GENECHIP GENOTYPING ANALYSIS SOFTWARE (GTYPE) Affymetrix GeneChip® Genotyping Analysis Software (GTYPE) is part of the GeneChip Mapping Array System, specifically designed to give highly accurate, automated SNP allele calls for GeneChip Mapping Arrays. In addition, GTYPE was developed to enable workflows for a variety of SNP-related applications, such as whole-genome association, linkage, and copy number studies. GTYPE provides high-quality genotyping, quality control reporting, and streamlined data analysis for projects with thousands of samples. GTYPE integrates with the BRLMM genotyping algorithm; BRLMM performs multiplechip analysis, fitting probe effects to increase precision on signal estimates for the two alleles of each SNP. This is followed by a Bayesian classification approach to make genotype calls. Learn more about BRLMM, the genotype calling algorithm, at www.affymetrix.com/support/technical/product_updates/brlmm_algorithm.affx Watch a video with Greg Marcus, Affymetrix Sr. Product Manager, discussing the algorithm improvements implemented in BRLMM at www.affymetrix.com/userForum/news/collaborations/marcus.uf Look at the GTYPE Data Analysis Workflow at www.affymetrix.com/support/technical/other/gtyp e_workflow_diagram.pdf GENECHIP-COMPATIBLE™ SOFTWARE 1.0 IMPACT OF NUMBER OF CONTROLS ON GENETIC POWER 0.6 0.4 0.2 Control: Case=1:1 Control: Case=2:1 Control: Case=3:1 Control: Case=4:1 0.0 Power 0.7 20% Increase 0.0 0.2 0.4 r2 0.6 0.8 1.0 Over 25 companies have built software that is integrated with the GeneChip system. If you tend to “buy” rather than “build” software, Affymetrix has already taken the first step to help you qualify your software vendors. Affymetrix’ GeneChipcompatible™ Application Program features commercial software vendors that have integrated their software products to work cleanly with the Affymetrix platform. The GeneChip-compatible Software Solutions Catalog (www.affymetrix.com/products/ software/compatible/snp.affx) provides a comprehensive list of software packages both at the level of SNP analysis as well nn nn 3 as laboratory management. Simply look for the GeneChip-compatible logo on any software package that you buy. GeneChip-compatible providers for SNP Analysis: · Golden Helix’ HelixTree® Genetics An a l y si s So f tw a r e View a recent GeneChip-compatible™ webcast illustrating a complete analysis workflow of data from the Mapping 500K Array Set at www.affymetrix.com/corporate/events/processEventRegistration Archive.jsp?event=71 · Progeny Software’s Progeny Lab View a recent webcast illustrating how Progeny Lab can aid in data management from the GeneChip Human Mapping 500K Array Set. www.affymetrix.com/corporate/events /processEventRegistrationArchive.jsp ?event=68 · Sapio Sciences’ Exemplar Genotyping Analysis Suite · Biocomputing Platforms’ BC/GENE JMP® Genetics from SAS The Platform PERFORM YOUR RESEARCH ON A PROVEN, STANDARDIZED PLATFORM The Mapping 500K Array Set is being used by over 200 investigators worldwide. There have been nearly 120 scientific publications in peer-reviewed journals, across multiple applications based on Affymetrix genotyping technology—including ground-breaking association studies in sudden cardiac death (Arking, et al.), obesity (Christman, et al.) and macular degeneration (Klein et al). Read over 120 Peer-reviewed Publications using the Mapping Assay at www.affymetrix.com/support/technical/other/mapping_publications_august _2006.pdf Watch an interview with Johns Hopkins School of Medicine’s, Aravinda Chakravarti whose team has identified a genetic variant associated with electocardiographic QT interval, a trait associated with increased risk of sudden cardiac death at www. microarraybulletin.com/community/article.php?p=2 41 OBESITY Watch an interview with Boston University Medical Center’s Alan Herbert and Michael Christman on their discovery of a gene that predisposes individuals to obesity at www.microarraybulletin.com/ community/article.php?p=133 The Integrated Genome Browser (IGB, pronounced “ig-bee”) from Affymetrix allows researchers to visualize genomic data and annotations from multiple data sources. IGB enables researchers to integrate genotyping and expression data into a single view of the genome, on a single platform. Read an interview with Harvard University School of Public Health’s Christoph Lange on algorithm development that enabled this study at www.microarraybulletin.com/community/article.ph p?p=206 AFFYMETRIX GENECHIP ® COMMAND CONSOLE ™ nn nn 4 GENOTYPING Fine Mapping/Replication The Custom Genotyping Assay 50,000 CUSTOM SNPS AT YOUR FINGERTIPS SUDDEN CARDIAC DEATH INTEGRATE EXPRESSION AND GENOTYPING DATA Affymetrix has announced the upcoming release of the next generation of instrument control software—Affymetrix GeneChip® Command Console™ (AGCC), which will co-exist with GCOS. Command Console software will leverage the latest software development technologies, including web-based user interfaces, file indexing, and integration via web services. Licenses to Command Console software will be freely available to all current Affymetrix customers. Learn more at: www.affymetrix.com/userForum/news/new Products/GC_CommandConsole.uf tics (IVD) use, providing a standardized platform for nucleic acid diagnostics. Additionally, Affymetrix will soon be the only microarray manufacturer to offer a CLIA-approved laboratory for development of clinical assays and samples. Under the Powered by Affymetrix™ program, companies can license technology from Affymetrix to develop innovative microarray products. The Mapping 500K Array Set is also being used by many major pharmaceutical companies conducting pharmacogenomics-based target discovery and clinical development. Read an interview with Allen Roses, SVP of Genetics at GlaxoSmithKline, who is conducting pharmacogenomic studies in multiple steps of drug discovery and development at www.microarraybulletin.com/community/article.php?p=242. DIAGNOSTICS Plan a diagnostic path for your study, and your business. Affymetrix has the first and only microarray instrument to be cleared by the FDA for in vitro diagnos Until recently, researchers have been pursuing just a handful of SNPs in the replication or fine mapping phase of an association study because there was no high-throughput, affordable technology that allowed them to genotype a customized set of SNPs. By applying a robust replication technology, the initial discovery scan can be maximally informative. With Affymetrix Custom Targeted Genotyping Panels, you no longer need to let technology limit your study. Using this system, which is based on Molecular Inversion Probe Technology, you can genotype up to 50,000 SNPs of your choice, enabling large-scale fine mapping and replication studies (Figures 4, 5, and 6). THE TARGETED GENOTYPING ASSAY Affymetrix Custom Targeted Genotyping allows you to genotype more of the SNPs you want. The conversion rate specification for the assay (the percentage of working SNPs that make it into the final assay panel from what was submitted) is greater than 80 percent, and typically greater than 90 percent. This conversion rate is considerably higher than other custom SNP genotyping assays on the market. Guarantees from other companies about conversion rates are based on the number of SNPs that have already passed a bioinformatic screening step. JUVENILE DIABETES Watch an interview with John Todd, from the JDRF/Wellcome Trust Diabetes Laboratory at www.microarraybulletin.com/ community/article.php?p=203 BOVINE TRAITS Watch an interview with Baylor College of Medicine’s Richard Gibbs at www.microarraybulletin. com/communit y/article.php? p=213 Figure 5: Affymetrix Targeted Genotyping Custom Panel Order Process. An Affymetrix Project Manager is assigned to assist you with your custom panel project. GENOTYPING SERVICES There is one protocol for all custom assay panels—from 3,000 to 50,000 SNPs; the assay leverages routine laboratory techniques, materials, and equipment. Conversion Rate: >80 percent Data Completeness: ³97.0 percent Repeatability: ³99.3 percent Accuracy: ³99.3 The Custom Genotyping Platform, Services, and Throughput You will be required to submit your gene list or SNP list by either CD or email. A preliminary QC is performed and the list of SNPs is returned for customer approval. Affymetrix will only make your panel when you are happy that the SNPs you want are included. Custom Panel Manufacturing Process CUSTOM ASSAY SPECIFICATIONS Researchers can perform targeted genotyping on their existing Affymetrix system with a simple upgrade, or have a service provider do the work. Genotyping services offer researchers a way to complete their projects quickly—simply send the samples in and receive data back. Affymetrix can provide this service, or researchers can work with one of our expert service providers— they have years of experience, extensive scientific expertise, and tight process controls. Affymetrix works with scientists to determine whether an in-house system or a service provider will best meet their needs. On your approval, Affymetrix will manufacture your custom panel. Approximately 8-12 weeks GET A STREAMLINED WORKFLOW Custom Panel Design Process Every Affymetrix Custom Targeted Genotyping oligo panel undergoes functional quality checks before it ever leaves our facility. Additionally, only Affymetrix Custom Targeted Genotyping allows you to see raw data from all four possible genotype calls. This gives researchers a built-in quality check for each SNP—you will know a SNP call has failed if there is too much background signal. With allele-specific probes, a researcher will not know that is the case. Approximately 2-4 weeks QUALITY CHECKS EVERY STEP OF THE WAY Your project manager will keep you updated with the status of your custom panel. Your custom panel product is delivered. THROUGHPUT THE PLATFORM Buy a standardized platform that’s proven in the field. The Affymetrix Custom Targeted Genotyping Assay has already been used to discover genetic associations in common diseases such as juvenile diabetes. The Affymetrix Targeted Genotyping Assay genotypes up to 50,000 SNPs* in 48 samples per day (including controls) and 192 samples per week. That is 2.4 million genotypes/day (Figure 7). Throughputs can be even higher by using additional equipment. Figure 4: Custom Targeted Genotyping combines custom SNP assay panels with Universal Tag Arrays. Custom SNP Assays You can genotype 3,000 to 50,000 SNPs on the same system and the same type of assay. Universal Tag Array Custom assays don’t require custom arrays. Universal Tag Arrays contain novel, bioinformatically designed tag sequences that result in minimal potential for crosshybridization and do not require redesign or recreation of custom GeneChip arrays. Custom 3K SNP Kit Universal 3K Array Custom 5K SNP Kit Universal 5K Array Custom 10K SNP Kit Universal 10K Array Custom 20K SNP Kit Universal 25K Array Custom 50K SNP Kit* Universal 70K Array *50K services only Custom Genotyping Software GET HIGHLY ACCURATE GENOTYPE CALLS The Affymetrix GeneChip ® Targeted Genotyping Analysis Software (GTGS) streamlines sample tracking and data analysis for Custom Targeted Genotyping. GTGS implements an enhanced genotype calling algorithm that improves the robustness and quality of genotyping. The expectation-maximization (E-M) algorithm handles the enormous number of data points (105-108) that need to be analyzed in a typical targeted genotyping experiment. It requires no manual intervention at the detailed level of a single call, sample, or marker. The algorithm performs data normalization followed by the application of a clustering algorithm for genotype calling. Optional marker filters are then applied to the clustered data, and per- nn nn 5 formance metrics, including conversion rate, call rate, repeatability, data completeness, and Mendelian consistency (if pedigree information is supplied) are reported. IMPROVE THE QUALITY OF YOUR GENOTYPES BY INSPECTING MORE OF YOUR DATA AT ONCE The GTGS software can genotype up to 5,000 samples in one batch. Genotypes for each marker are determined only after inspecting the distribution of signal intensities for all samples in the batch. Inspection of genotypes across samples improves genotyping quality. Rare alleles, like those in green, are more easily detected when more samples can be analyzed at one time (Figure 8). Figure 6: The Custom Targeted Genotyping System is based on the Molecular Inversion Probe Assay. Assay Panel 3K probes 5K probes 10K probes 20K probes 50K probes Genomic DNA WGA DNA Mix DNA and assay (SNP) panel together Aliquot into four reactions to be queried by different dNTPs Identify the nucleotide at each assay locus (SNP locus) using Universal Tag Array Label each aliquot with a different color MAKE QUALITY CONTROL CHECKS MORE EFFICIENT Quality control (QC) is streamlined with GTGS because the software automatically flags samples or markers that do not pass quality control criteria. GTGS quality checks on three levels: for each call individually (signals from all four nucleotides are measured), at the sample level (is the overall data quality from a single array sufficient?), and at the marker level (is the data quality for a marker across multiple samples sufficient?). GTGS provides a visualization of each QC step, such as trend charts to look at QC metrics vs. time, and sample location, as well as a visual inspection of each array at each nucleotide channel. TRACK YOUR SAMPLES FROM START TO FINISH The complete custom genotyping assay is divided into two major steps: pre-amplification and post-amplification. Dedicated laboratory space and computer stations are recommended for both steps. The GTGS software allows you to track your samples from sample plate to GeneChip array between these laboratory areas. EXPORT YOUR DATA IN ONE OF MULTIPLE TEXT-BASED FORMATS Genotypes are exported as a single file, collating the data across all of your samples belonging to a single-batch genotyping operation. The tab-delimited output provides the flexibility to reformat as desired. nn nn 6 GENOTYPING Figure 7: Researchers can genotype up to 192 samples per week with Affymetrix Custom Targeted Genotyping. That’s 2.4 million genotypes per day. Monday Tuesday Wednesday Thursday Friday Anneal 48 Samples (for Tuesday) Anneal 48 Samples (for Wednesday) Anneal 48 Samples (for Thursday) Anneal 48 Samples (for Friday) Hyb 48 Samples (from last Friday for Tuesday) Process & Hyb 48 Samples (for Wednesday) Process & Hyb 48 Samples (for Thursday) Process & Hyb 48 Samples (for Friday) Process & Hyb 48 Samples (for next Monday) Scan 48 Samples (from Monday) Scan 48 Samples (from Tuesday) Scan 48 Samples (from Wednesday) Scan 48 Samples (from Thursday) Per Day: Per Week: - 20K Assay Panel - 46 Samples (48 - 2 Controls) - 46 Samples x 20,000 Genotypes/Sample ~ 1 Million Genotypes/Day - 20K Assay Panel - 192 Samples (does not include controls) - 192 Samples x 20,000 Genotypes/Sample ~ 3.8 Million Genotypes/Day Resequencing The Affymetrix CustomSeq® Resequencing Program The Affymetrix CustomSeq® Resequencing Program enables researchers to design custom arrays for rapid identification of all SNPs—common, rare, or non-synonymous—in a specific genomic region. Complete sequence analysis ensures the highest probability of finding causative SNPs, since many causal variants are rare or heterogeneous in the population. CustomSeq Arrays provide a highthroughput method of SNP detection by enabling the analysis of large contiguous chromosomal regions, or multiple candidate genes, on a single array. with an interview Read University of Connecticut’s, Linda Strausbaugh on array-based resequencing. www.microarraybulletin.com/community/article. php?p=67 Figure 8: A screen shot of the GeneChip® Targeted Genotyping Analysis Software (GTGS). ® EXPERIMENT SUMMARY: · Sequenced 155 exons from 35 patients (29,214 bp) from genes associated with autosomal recessive Retinitis Pigmentosa (arRP), a condition leading to irreversible blindness or severe visual impairment, and affecting one in 3500 individuals worldwide · Retinitis Pigmentosa shows broad genetic heterogeneity with at least 32 genes known to be associated with various forms (autosomal dominant, autosomal recessive, and X-linked) BASE CALLING PERFORMANCE · Average Call Rates=97.60 percent (individual arrays ranged from 96.0 percent to 98.5 percent) · Accuracy >99 percent · Reproducibility >99 percent SEQUENCE MORE DNA FOR LESS THAN A TENTH OF A PENNY PER BASE CustomSeq Arrays enable the analysis of up to 300,000 bases of double-stranded sequence (600,000 bases total) on a single array, providing the most efficient and cost-effective method for interrogating large amounts of sequence in a single experiment. GET HIGH-QUALITY SEQUENCING DATA IN A SINGLE PASS Affymetrix CustomSeq Arrays call greater than 90 percent of bases at greater than 99.9 percent accuracy and 99.9 percent reproducibility. PERFORMANCE ON CEPH SAMPLES Affymetrix has characterized the performance of the 300kb resequencing arrays across multiple designs, representing homozygous and heterozygous model systems. To demonstrate performance for a typical human region, resequencing array data and dideoxy sequence data were collected from 16 diploid CEPH (Centre d’etude du polymorphisme humaine) individuals across 115kb of non-repetitive sequence on Chromosome 4—1.84 mega base pairs (Mb) in total. While the performance of every customdesigned array depends on the specific genomic sequence, the overall call rate in this study was found to be 96.56 percent; overall accuracy was 99.95 percent. CUSTOMSEQ ARRAY THROUGHPUT CustomSeq Arrays give you data faster, so you can find your SNPs faster. CustomSeq arrays deliver completed sequence in 48 hours with minimal alignment, curation, or hand editing. Put your time and money into your experiments, not your reagents. By leveraging long-range PCR, the number of amplification reactions required can be dramatically reduced, decreasing the cost and time associated with PCR. Do your resequencing, whole-genome scanning, and targeted genotyping on one reliable, integrated Affymetrix system. CUSTOM RESEQUENCING PUBLICATIONS Take advantage of a proven, cutting-edge technology. The publications below highlight the advantages and system performance in two candidate gene studies. 1. Sequencing Arrays for Screening Multiple Genes Associated with EarlyOnset Human Retinal Degenerations on a High-Throughput Platform. Mandal MN, et al . Invest Opthalmol Vis Sci. 46 (9):3355-62(2005). SNP DETECTION · 506 sequence changes identified · Accurately detected 382 previously reported SNPs and identified 113 novel SNPs · Accurately detected five previously reported mutations and identified seven novel rare mutations 2. A Transforming MET Mutation Discovered in Non-small Cell Lung Cancer Using Microarrray-based Resequencing. Tengs T., et al. Cancer Lett. (2005). EXPERIMENT SUMMARY: · Sequenced 164 exons (23,966 bp) from genes associated with cancer in 20 lung tumor samples with matched normal controls · Dideoxy sequencing was performed on a subset of exons in order to evaluate the performance of the arrays COMPARISON TO DIDEOXY SEQUENCING · Call rate=97.53 percent · Overall accuracy=99.99 percent · Only four SNP call errors reported · Three homozygous SNPs called heterozygous and one heterozygous SNP called reference nn nn 7 Functional Tests Figure 9: Applications for Affymetrix Expression Products. Functional Testing Through Expression Analysis Finding a causal variant or gene is just the beginning—understanding how that affects the entire biological system is the critical next step to ultimately developing a therapeutic or diagnostic. A new generation of expression microarrays allows you to examine all aspects of the genome— exons, introns, and other non-coding regulatory regions—to decipher pathways and functional effects. GeneChip® Exon Arrays enable you to identify expression profiles affected by a disease-causing variant or gene, including altered splicing patterns. GeneChip® Tiling Arrays for ChIP-on-chip analysis allow you to identify regions of DNA with which your specific protein interacts—at the highest resolution, and with the most complete coverage of the human genome. Affymetrix 3'IVT Expression Arrays are the industry standard and have been used in thousands of studies. AFFYMETRIX, INC. AFFYMETRIX UK Ltd AFFYMETRIX JAPAN K.K. 3420 Central Expressway Santa Clara, CA 95051 USA Tel: 1-888-DNA-CHIP (1-888-362-2447) Fax: 1-408-731-5441 [email protected] [email protected] Voyager, Mercury Park, Wycombe Lane, Wooburn Green, High Wycombe HP10 0HH United Kingdom UK and Others Tel: +44 (0) 1628 552550 France Tel: 0800919505 Germany Tel: 01803001334 Fax: +44 (0) 1628 552585 [email protected] [email protected] Mita NN Bldg., 16 F 4-1-23 Shiba, Minato-ku, Tokyo 108-0014 Japan Tel: +81-(0)3-5730-8200 Fax: +81-(0)3-5730-8201 [email protected] [email protected] www.affymetrix.com Please visit our web site for international distributor contact information. For research use only. Not for use in diagnostic procedures. Part No. 702256 Rev. 1 © ® 2006 Affymetrix, Inc. All rights reserved. Affymetrix®, , GeneChip®, HuSNP®, GenFlex®, Flying Objective™, CustomExpress®, CustomSeq® , NetAffx™, Tools To Take You As Far As Your Vision®, The Way Ahead™, Powered by Affymetrix™, GeneChip-compatible™, and Command Console™ are trademarks of Affymetrix, Inc. Products may be covered by one or more of the following patents and/or sold under license from Oxford Gene Technology: U.S. Patent Nos. 5,445,934; 5,700,637; 5,744,305; 5,945,334; 6,054,270; 6,140,044; 6,261,776; 6,291,183; 6,346,413; 6,399,365; 6,420,169; 6,551,817; 6,610,482; 6,733,977; and EP 619 321; 373 203 and other U.S. or foreign patents.