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Current Genetic Analysis Technology Lee Murphy Wellcome Trust Clinical Research Facility

Current Genetic Analysis Technology Lee Murphy Wellcome Trust Clinical Research Facility. WTCRF. Nursing & Clinical Epidemiology & Statistics Image Analysis. www.wtcrf.ed.ac.uk. WTCRF. Good Clinical Practice & the EU Directive Statistics for clinical researchers

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Current Genetic Analysis Technology Lee Murphy Wellcome Trust Clinical Research Facility

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  1. Current Genetic Analysis TechnologyLee MurphyWellcome Trust Clinical Research Facility

  2. WTCRF • Nursing & Clinical • Epidemiology & Statistics • Image Analysis www.wtcrf.ed.ac.uk

  3. WTCRF • Good Clinical Practice & the EU Directive • Statistics for clinical researchers • How to fill out an ethics form • Good Laboratory Practice www.wtcrf.ed.ac.uk/education/courses.htm

  4. Platforms ABI 7900 Illumina Beadstation Illumina Genome Analyser 1-30 SNPs 96-3,000 SNPs 1bp to >1Gbp (Goldengate) 1,000’s samples 6,000-1,000,000 SNPs 8 samples at a time (Infinium) 96 samples at a time

  5. Collection of blood & clinical data (“phenotype”) from subjects Extraction &archivingof DNA Analysis of sequence variation (“genotype”) Genotype : Phenotype correlation C→T

  6. Study Design The number of cases and controls required for adequate study power. Cases and controls should be randomized between plates. Decide on the DNA and sample exclusion criteria Replicating any positive association results using a different chemistry and platform such as Applied Biosystems Taqman assays or DNA sequencing. Replication in an independent population to confirm any positive association results. McCarthy, M. et al. (2008) Genome-wide association studies for complex traits: consensus, uncertainty and challenges. Nature Reviews Genetics9: 356-369. NCI-NHGRI Working Group on Replication in Association Studies (2007) Replicating genotype–phenotype associations. Nature447: 655-660.

  7. Sample Size See your statistician Always more than you think Sample size packages Quanto Genetic Power Calculator Need to know/estimate lots of information Prevalence Interactions Clinical assessment & genotyping errors Burton, P. et al. (2008) Size matters: just how big is BIG? Int. J. Epiodemiol.

  8. Control DNA GS:Donor DNA Databank ethically approved control DNA set from blood donors in Scotland. Plasma and short questionnaire data also collected. 4,999 DNA samples www.generationscotland.org

  9. Control DNA

  10. Platforms ABI 7900 Illumina Beadstation Illumina Genome Analyser 1-30 SNPs 96-3,000 SNPs 1bp to >1Gbp (Goldengate) 1,000’s samples 6,000-1,000,000 SNPs 8 samples at a time (Infinium) 96 samples at a time

  11. ABI 7900 – Taqman Genotyping

  12. Detection of Bi-allelic Variation A Homozygous for A A T Homozygous for T T A Heterozygous T

  13. A R Q T R Q A A R Q T R Q R Q Q R

  14. Taqman Genotyping

  15. Illumina Microarray

  16. Illumina Genotyping Infinium (7,600-1 Million SNPs) HumanCNV370-Quad Human610-Quad Human1M-Duo iSelect Custom Genotyping GoldenGate (96-1,536 SNPs) Mouse & Linkage Panels Cancer SNP Panel GoldenGate Custom Genotyping

  17. Infinium Single-Base Extension

  18. Infinium Genotype Calling

  19. Copy Number Variation Macciardi 2008 Redon, R. et al. (2006) Global variation in copy number in the human genome. Nature444: 444-454. Carter, N. (2007) Methods and strategies for analyzing copy number variation using DNA microarrays. Nature Genetics 39: S16-S21

  20. Next-Generation Sequencers Roche FLX Illumina Genome Analyser ABI Solid genepool.bio.ed.ac.uk

  21. Illumina Genome Analyser www.Illumina.com

  22. www.Illumina.com

  23. Next-Next-Generation Sequencers • www.helicosbio.com • 100-200bp read lengths • 1billions reads/day • www.pacificbiosciences.com • >10,000bp read lengths • $100 genome in an hour Single Molecule Sequencing - Towards the $1,000 Genome in One Day

  24. Acknowledgements WTCRF – Genetics Core Mark Blanford Richard Clark Audrey Duncan Louise Evenden Angie Fawkes Jude Gibson William Hawkins Julie Morrison Lee.Murphy@ed.ac.uk 0131 537 3370 www.wtcrf.ed.ac.uk/genetics/

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