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 How to fill out an ethics form Good Laboratory Practice www.wtcrf.ed.ac.uk/education/courses.htm

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

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

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. Exclude samples with >10% missing SNP Exclude SNPs with <90% call rate McCarthy, M. et al. (2008) Genome-wide association studies for complex traits: consensus, uncertainty and challenges. Nature Reviews Genetics 9: 356-369. NCI-NHGRI Working Group on Replication in Association Studies (2007) Replicating genotype–phenotype associations. Nature 447: 655-660.

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 Test reliability: BMI = >0.95 Hip/waist circumference = 0.9 Blood pressure = 0.7 Diet recall = 0.3-0.5 Burton, P. et al. (2008) Size matters: just how big is BIG? Int. J. Epiodemiol.

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

Control DNA Region % Female % Male All GS:3D (%) 2001 Census Strathclyde 48.04 48.22 48.14 43.63 Lothian 14.67 14.52 14.58 18.24 Tayside 4.26 4.18 4.22 7.68 Fife and Central 12.78 11.39 12.01 9.56 Grampian 7.58 8.67 8.19 10.39 Borders 3.38 3.61 3.50 2.11 Highland (mainland) 3.76 3.48 3.60 4.13 Highland (islands) 2.38 1.94 2.13 1.34 Dumfries & Galloway 3.15 4.00 3.63 2.92

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

ABI 7900 – Taqman Genotyping

Detection of Bi-allelic Variation Homozygous for A A T Homozygous for T T SNP of interest A Heterozygous T

R A Q R T Q A R A Q R T Q R Q R Q

Taqman Genotyping

Illumina Microarray

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

Infinium Single-Base Extension

Infinium Genotype Calling Same principal to taqman – 3 clusters

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

Next-Generation Sequencers Roche FLX Illumina Genome Analyser ABI Solid Platform Read Length Total Output Roche FLX 200-300bp 100Mb Illumina Genome Analyser ~35bp 3000Mb ABI Solid ~35 6000Mb genepool.bio.ed.ac.uk

Illumina Genome Analyser www.Illumina.com

www.Illumina.com

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

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/