1. Current Genetic Analysis Technology Lee Murphy Wellcome Trust Clinical Research Facility

2. WTCRF Nursing & Clinical Epidemiology & Statistics Image Analysis

3. WTCRF Good Clinical Practice & the EU Directive
Statistics for clinical researchers
How to fill out an ethics form
Good Laboratory Practice

4. Platforms ABI 7900 Illumina Beadstation Illumina Genome Analyser
1-30 SNPs ,000 SNPs 1bp to >1Gbp
(Goldengate)
1,000’s samples ,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 & archiving of 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.
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:
NCI-NHGRI Working Group on Replication in Association Studies (2007) Replicating genotype–phenotype associations. Nature 447:

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
Test reliability:
BMI = >0.95
Hip/waist circumference = 0.9
Blood pressure = 0.7
Diet recall =
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

9. 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

10. Platforms ABI 7900 Illumina Beadstation Illumina Genome Analyser
1-30 SNPs ,000 SNPs 1bp to >1Gbp
(Goldengate)
1,000’s samples ,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
Homozygous for A A T
Homozygous for T T
SNP of interest A
Heterozygous T

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

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

19. Infinium Genotype Calling
Same principal to taqman – 3 clusters

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

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

22. Illumina Genome Analyser

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

25. Acknowledgements WTCRF – Genetics Core Mark Blanford Richard Clark
Audrey Duncan
Louise Evenden
Angie Fawkes
Jude Gibson
William Hawkins
Julie Morrison