MICROARRAYS+SEQUENCING

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1 MICROARRAYS+SEQUENCING The most efficient way to advance genomics research Down to a Science.

2 Affymetrix GeneChip Expression Technology Complementing your Next-Generation Sequencing (RNA-Seq) Experiments As next-generation sequencing (NGS) of cdna (RNA-Seq) becomes more widely adopted for transcriptome profiling, Affymetrix microarrays are an increasingly indispensible complement to these emerging approaches. As with all new technologies, RNA-Seq studies present significant challenges, including sample input requirements and the complex, voluminous data generated. Utilizing Affymetrix microarrays in your transcriptome profiling studies allows you to: Quickly and cost effectively generate comprehensive datasets across known pathways and genes, enabling you to focus time-consuming RNA-Seq experiments on those studies that require the discovery of unknown transcription Perform global gene expression profiling with very limited amounts of RNA or degraded FFPE RNA not amenable to RNA-Seq Better quantify low abundance transcripts Validate many RNA-Seq hits simultaneously Develop a clinical test using an FDA-approved platform...the decision of whether to use DNA microarrays or RNA-Seq seems straightforward and unambiguous. In reality, the two technologies couldn t be more complementary. Scott Peterson JCVI Weblog J. Craig Venter Institute

3 Affymetrix microarrays for pathway and gene profiling studies RNA-Seq for identification of unknown transcription Understand changes in gene expression of known pathways and regulatory genes responsible for a biological process or disease phenotype with Affymetrix microarrays. Given the extensive expression information already discovered through the efforts of scientists over the last 15 years, the regulatory pathways and most important regulatory genes have been identified and exist on current generation GeneChip Arrays. Achieve biologically relevant results cost effectively, easily, and quickly (within 3 days) by using well-established software solutions requiring no particular bioinformatics expertise Perform your RNA-Seq experiments for studies in which the identification of transcripts not yet in the public domain is critical Figure 1 Gene expression changes identified with microarrays in embryonic stem cells during differentiation. 1 Figure from GenMapp:

4 Gene expression profiling from FFPE samples In translational research Often, RNA collected from valuable study samples is either very limited in amount or of poor quality (such as RNA isolated from FFPE samples). Currently, RNA-Seq protocols for preparing RNA of limited quantity or poor quality are either unavailable or not validated. Collect robust gene expression data from Affymetrix microarray experiments using very limited quantities of RNA (down to tens of picograms) and degraded RNA from precious FFPE samples with FFPE optimized arrays, reagents, and protocols. More than 50 publications from the last 18 months prove that Affymetrix microarrays generate valuable expression data from these challenging samples. (See referenced publications 2 8 on page 8.) Figure 2 FFPE samples used to identify signature to sub-group diffuse, large B-cell Lymphoma samples. 2 Signature FFN (GA) FPET (GA) Biological pathways: IPA (list comparisons) log(p-value) Threshold 0.0 Cellular Development Hematological System Development and Function Cell Cycle Cancer Hematological Disease Cell Death Cellular Growth and Proliferation Immune Response Immune and Lymphatic System Development and Function Immunological Disease Tumor Morphology Results of pathway analysis from Ingenuity IPA Software. The horizontal gold bar indicates the threshold of significance. The signature pathways listed in darkest blue come from the list of genes (published by Wright, et al 19 ) that separate ABC from GCB subgroups.

5 Gene expression quantitation of low-abundance transcripts Due to the nature of RNA-Seq quantitation, the precision of RNA quantitative measurements is dependent on the abundance of that transcript; in other words, the less abundant the transcript, the fewer the number of reads and therefore the higher the variability in the quantitative measurement. 9,10 Affymetrix microarrays provide consistent, precise gene expression quantitation regardless of transcript abundance. Figure 3 Coefficient of variance (CoV) of measured expression levels of exons and genes with RNA-Seq (39 million mapped reads) and Affymetrix microarrays. 11

6 Validation of RNA-Seq results with microarrays Validate your RNA-Seq results with Affymetrix microarrays by simultaneously measuring thousands of potential transcript changes quickly and easily. While PCR techniques require selection of a limited number of transcripts for confirmation, the portfolio of Affymetrix expression microarrays offers simple robust, cost-effective options. Figure 4 Confirmation of RNA-Seq expression changes with Affymetrix microarrays. 12 Biomarker validation and clinical test development Clinically validate your expression signature and develop your test with FDA proven technology. Affymetrix has FDA-approved instruments and reagents that have been used in a number of clinical tests. Affymetrix Arrays have been used in an FDA-approved expression-based test, including the use of RNA isolated from FFPE samples. Pathwork Diagnostics ( provides an FDA-approved Affymetrix GeneChip expression array-based diagnostic: the Tissue of Origin Test. 13 In 2011, Pathwork Dx expanded the applicability of this approved test by allowing the use of RNA isolated from FFPE tissue sources, demonstrating the applicability of FFPE expression-based tests for use in clinical testing (Figure 5). 14

7 Tissue Similarity score Similarity score Colorectal Pancreas Non-small cell lung Breast Gastric Kidney Hepatocellular Ovarian Soft tissue sarcoma Non-Hodgkin s lymphoma Thyroid Prostate Melanoma Bladder Testicular germ cell LOW low HIGH Figure 5 FFPE samples to report for Pathwork Diagnostics Tissue of Origin Test. 14 To complement your NGS studies, Affymetrix GeneChip Gene Expression Solutions are: Cost effective: with a variety of GeneChip Expression Array options Robust: generating high-quality data from standard, degraded RNAs (FFPE) and limited RNA quantities (down to tens of picograms) Stable: with mature and fully commercialized and optimized workflows (manual and automated), including software analysis solutions Quick: generating publishable results in days Comprehensive: containing all known pathways, genes, and transcripts Proven: more than 20,000 gene expression publications demonstrating the value of the technology with several FDA-approved tests on the market

8 References Williams P. M., et al. A novel method of amplification of FFPET-derived RNA enables accurate disease classification with microarrays. The Journal of Molecular Diagnostics 12(5): (2010). 3 Burlington B., et al. CD40 pathway activation status predicts response to CD40 therapy in diffuse large B cell Lymphoma. Science Translational Medicine 3(74):74ra22 (2011). 4 Gravendeel L. A., et al. Gene expression profiles of gliomas in formalin-fixed paraffin embedded material. British Journal of Cancer 106(3): (2012). 5 Linton K., et al. Microarray gene expression analysis of Fixed archival tissue permits molecular classification and identification of potential therapeutic targets in diffuse large B-Cell lymphoma. Journal of Molecular Diagnostics 14(3): (2012). 6 Volkmer J. P., et al. Three differentiation states risk-stratify bladder cancer into distinct subtypes. Proceedings of the National Academy of Sciences 109(6): (2012). 7 Kennedy R. L., et al. Development and independent validation of a prognostic assay for stage II colon cancer using formalin-fixed paraffin-embedded tissue. Journal of Clinical Oncology 29(35): (2011). 8 Hall J. S., et al. Exon-array profiling unlocks clinically and biologically relevant gene signatures from formalin-fixed paraffin-embedded tumour samples. British Journal of Cancer 104(6): (2011). 9 Jiang L., et al. Synthetic spike-in standards for RNA-Seq experiments. Genome Research 21(9): (2011). 10 Labaj P. P., et al. Characterization and improvement of RNA-Seq precision in quantitative transcript expression profiling. Bioinformatics 27(13):i383 i391 (2011). 11 Xu W., et al. Human transcriptome array for high-throughput clinical studies. Proceedings of the National Academy of Sciences 108(9): (2011). 12 Hackett N. R., et al. RNA-Seq quantification of the human small airway epithelium transcriptome. BMC Genomics 13:82 (2012). 13 Monzon F. A., et al. Multicenter Validation of a 1,550-Gene Expression Profile for Identification of Tumor Tissue of Origin. Journal of Clinical Oncology 27(15): (2009). 14 Pillai R., et al. Gene Expression Test for Tumor Identification in Formalin-Fixed, Paraffin-Embedded Specimens. Journal of Molecular Diagnostics 13(1):48-56 (2011). World-class support Affymetrix offers an expanding portfolio of customer support and services from training and instrument maintenance to consulting and compliance led by our world-class team of multilingual technical experts, field application scientists (FAS), and regional field service engineers (FSE). For more information please visit Affymetrix, Inc. Tel: Affymetrix UK Ltd. Tel: +44-(0) Affymetrix Japan K.K. Tel: +81-(0) Panomics Solutions Tel: panomics.affymetrix.com USB Products Tel: usb.affymetrix.com Please visit our website for international distributor contact information. For Research Use Only. Not for use in diagnostic procedures. P/N FM01567 Rev. 1 Affymetrix, Inc. All rights reserved. Affymetrix, Axiom, Command Console, CytoScan, DMET, GeneAtlas, GeneChip, GeneChip-compatible, GeneTitan, Genotyping Console, mydesign, NetAffx, OncoScan, Panomics, Powered by Affymetrix, PrimeView, Procarta, and QuantiGene are trademarks or registered trademarks of Affymetrix, Inc. All other trademarks are the property of their respective owners. Products may be covered by one or more of the following patents: U.S. Patent Nos. 5,445,934; 5,744,305; 5,945,334; 6,140,044; 6,399,365; 6,420,169; 6,551,817; 6,733,977; 7,629,164; 7,790,389 and D430,024 and other U.S. or foreign patents. Products are manufactured and sold under license from OGT under 5,700,637 and 6,054,270.