Transcript Illumina Microarray Seminar

Current Genetic Analysis
Technology
Lee Murphy
Wellcome Trust Clinical Research Facility
WTCRF
• Nursing & Clinical
• Epidemiology & Statistics
• Image Analysis
www.wtcrf.ed.ac.uk
WTCRF
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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
1-30 SNPs
1,000’s samples
Illumina Beadstation
96-3,000 SNPs
(Goldengate)
6,000-1,000,000 SNPs
(Infinium)
96 samples at a time
Illumina Genome Analyser
1bp to >1Gbp
8 samples at a time
Collection of blood & clinical
data (“phenotype”) from
subjects
Extraction & archiving
of DNA
Analysis of sequence
variation (“genotype”)
C→T
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
%
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
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
Region
Fife and Central
Platforms
ABI 7900
1-30 SNPs
1,000’s samples
Illumina Beadstation
96-3,000 SNPs
(Goldengate)
6,000-1,000,000 SNPs
(Infinium)
96 samples at a time
Illumina Genome Analyser
1bp to >1Gbp
8 samples at a time
ABI 7900 – Taqman Genotyping
Detection of Bi-allelic Variation
A
A
Homozygous for A
T
T
Homozygous for T
A
T
Heterozygous
R
A
Q
T
R
A
R
A
Q
R
R
R
T
Q
Q
Q
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
Read Length
Total Output
Roche FLX
200-300bp
100Mb
Illumina Genome ~35bp
Analyser
3000Mb
ABI Solid
6000Mb
~35
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/