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National Genotyping Center

National Genotyping Center. Barcelona, July 11, 2005. International Workshop on Pharmacogenomics and Pharmacogenetics. Limited therapeutic efficacy in many TAs. Biomedical R&D funding vs drug registration. Output of new drugs x 0.25. R&D investment x2.5.

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National Genotyping Center

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  1. NationalGenotyping Center Barcelona, July 11, 2005 International Workshop on Pharmacogenomics and Pharmacogenetics

  2. Limited therapeutic efficacy in many TAs

  3. Biomedical R&D funding vs drug registration Output of new drugs x 0.25 R&D investment x2.5 L.J. Lesko & J. Woodcock Nat Rev Drug Discov (2004)

  4. FINDING GENES THAT UNDERLIE COMPLEX TRAITS (QUANTITATIVE TRAIT LOCI OR QTLs) PHARMACOGENETICS/PHARMACOGENOMICS SNP mapping applications

  5. Association studies Candidate gene approach -Causative hypothesis or candidate genes -Genes can be too numerous -Bias towards specific genes Whole genome scans -No need of gene selection -Regions of genomes rather than genes -SNP density/Number of samples- Power of the studies

  6. The druggable genome

  7. Anatomy of Chromosome 6 LD Blocks Caucasian LD Blocks African LD Blocks HapMap and MapVar project

  8. Spanish National Genotyping Center Nodes: Santiago: SNPlex Sequenom Barcelona: SNplex Madrid: Illumina Budget: € 2-3 million/year 2003-2008 Infrastructure and personnel to participate in the International HapMap project and to carry out large scale association studies

  9. GeGen Scientific International Committee Ethical International Committeel Coordination Barcelona (UPF) Jaume Bertranpetit NODE 2 Santiago de Compostela (USC) NODE 1 Barcelona (CRG) (UPF) NODE 3 Madrid (CNIO) Xavier Estivill SNPlex / TaqMan Javier Benítez Illumina / TaqMan Angel Carracedo Sequenom / SNPlex/ TaqMan

  10. Santiago-USC • Director: Angel Carracedo • Supervisor: Chris Phillips • Manager Sequenom: María Torres • Manager SNPlex: Beatriz Sobrino • Bioinformatics: Jorge Amigo • Technicians: Inés Quintela, Begoña C., Carmen R • Project managers: • María Brión • (oftalmology, cardiovascular) • Xabi Costas • (psychiatry, neurology, inflamation) • Ana Vega-Toño Salas • (cancer, pop.genetics, anesthesia)

  11. Barcelona • Director: Xavier Estivill • Responsable plataforma: Mònica Bayés • Project Manager:Mònica Gratacòs • Data Manager: Rafael de Cid • Bioinformático:a determinar • Técnicos Carles Arribas Cecília García Magda Monfort Anna Puig

  12. Madrid Director: Javier Benitez Supervisor y Project Manager: Anna González-Neira Estadístico y Project Manager: Roger Milne Bioinformático: Jesús Lopez Técnicos: Emilio González Cristian Torrenteras

  13. Genotyping technologies Illumina SNPlex Sequenom TaqMan 1500 - 300 SNPs 400 - 40 SNPs 200- 5 SNPs 10 - 1 SNPs

  14. Automatedpreparation Targets Mass Spectrometry AutomatedAnalysis Robotics Chip MALDI-TOF Mass Spectrometer Analysis Software MALDI-TOF-MS + SBE DNA Preparation DNA Purification Rapid evolution from 5 plex average to 10 plex to 20 plex

  15. Analysis Software for checking data improved (good collaboration with the company)

  16. Prep gDNA Sample(s) Add gDNA To Ligation Mixture OLA in Thermal Cycler Clean up Unligated Probes Universal PCR Capture PCR Products Hyb. Drag Chutes Elute Drag Chutes & Load on CE Generate Raw Data Primary Analysis & QC of data Secondary Data Analysis Steps in Green are multiplexed SNPlex Assay- Workflow Overview S A M P L E P R E P TECAM robotics S A M P L E P R E P C E A N A L Y S I S ABI3730

  17. • • • SNPlex OLA/PCR Assay (AB) Drag Chutes Load on CE instrument Mobility Modifiers SNP 1 cZipcode • • • • ABI3730 SNP 2 • • •

  18. Hybridization to Universal IllumicodeTM Array Decoding by Sequential Hybridization

  19. Illumina Fiber Optic Bundles • Cy3 channel only • 3 micron diameter beads • 5 micron center-to-center spacing • 49,777 features on 1.4 mm diameter bundle • 1520 bead types • ~30 copies of each bead type

  20. 1152 Multiplex for 96 DNAs by Individual Bead • Rapid evolution from 384 > 768 > 1152plex

  21. Platforms • Illumina: Large scale projets-Whole Genome Scans • Sequenom (MALDITOF-MS): Small and medium size projects • SNPlex: Medium-Large scale genotyping projects • Decision on the platforms to be used depending on cost, time scale, characteristics of the project (i.e. whole genome scans, flexibility in SNP selection-conversion rate) (combining platforms is usually necessary)

  22. CeGen Coordinación • Pre-genotyping • DNA extraction and quantification • Project design • SNP selection based on: • Function prediction • Previous LD • Known disease association • Post-genotyping • Data download • Format changes • Data analysis • Customer support Genotyping platforms: Designs Genotyping Data checking Researchers Bioinformatics: Coordination: Arcadi Navarro UPF

  23. http://bioinfo.cnio.es/cgi-bin/lucia/pupasnp2/pupasnp2.cgi Validation status by-frequency by-2hit-2allele by-cluster Define Database Past your gene Flanking region Funcional properties Non synonymous TFBS (10000 bp upstream) Exonic Splicing Enhancer Intron Boundaries Mus Musculus conserved Type Coding Intro UTR Local Population frequency

  24. Genotyping Data Formatter (GDF) • Need: Checking results of genotyping output • Problem: Amount of data • Tools: • Genotyping output analysis • Cross-checking for error detection • Data ready-to-use for specific software analysis(Arlequin, Phase, …)

  25. miniLIMS • Share information between investigators • Problems: Different information input, different needs for data access, organization, structure, ... • Results: • Complete framework for project management • Personalized access to data

  26. Other developments • Data loading scripts for different genotyping platforms • Direct processing of data from platforms output • Re-sampling scripts for association studies • Bioinformatic implementation of classical and new mathematical tools for association

  27. Mathematical tools for complexity • Sample stratification analysis • Multivariate adaptive regression splines • Bayesian networks • Combinatorial-partitioning method • Classification and regression trees • Restricted partition method • Multifactor dimensionality reduction • Artificial neural networks

  28. National Genotyping Center 2005 • 4 pilot projects in 2004 • CeGen offered as service to the scientific community in Jan, 2005 • More than 20 projects • Psychiatry (7 projects >4M genotypes) • Rheumatology (5 projects >3M genotypes) • Cancer (3 projects>2M genotypes) • Ophtalmology (3 projects) • Cardiovascular (2 projects) • Neurology (2 projects) • Forensics (2 projects) • Pharmacogenetics (1 project) • Non-human (1 project)

  29. http://www.cegen.org

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