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

ABI 7900

Platforms

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

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

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 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 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

Strathclyde Lothian Tayside Fife and Central Grampian Borders Highland (mainland) Highland (islands) Dumfries & Galloway %

Female

48.04

14.67

4.26

12.78

7.58

3.38

%

Male

48.22

14.52

4.18

11.39

8.67

3.61

3.76

2.38

3.15

3.48

1.94

4.00

All GS:3D

(%) 48.14

14.58

4.22

12.01

8.19

3.50

3.60

2.13

3.63

2001 Census

43.63

18.24

7.68

9.56

10.39

2.11

4.13

1.34

2.92

ABI 7900

Platforms

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

ABI 7900 – Taqman Genotyping

Detection of Bi-allelic Variation

A A T T A T Homozygous for A Homozygous for T Heterozygous

R A Q A R T Q 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

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

Roche FLX

Read Length

200-300bp Illumina Genome Analyser ~35bp ABI Solid ~35

Total Output

100Mb 3000Mb 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 [email protected]

0131 537 3370 www.wtcrf.ed.ac.uk/genetics/