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Golden Gate technology an efficient technology for

performing Association studies with tag-SNPs on large cohort

M. Vanpeene, V. Decaulne, S. Roy,

P. Lindenbaum and F. RousseauIntegraGen SA, www.integragen.com

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Strategy

Molecular Diagnostics

Target Validation

Patient collections

Therapeutic Focus

Bi-polarDisorder

Obesity

Autism

Type 2Diabetes

Others

GenomeHIP™

IntegraCHIPtm

Access by IntegraGen

Access by Partner

Quicker, Faster, with more precision

Objectives: Development of Genetic Tests & Identification of Targets

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DNA fragmentation and addition of Flags

Denaturation and Mix DNA

Re-hybridize to form hybrid DNA molecules

LOCALISATION OF THE IBD REGION ON THE GENOME

Hybridisation on DNA chip (IntegraChip) representing the whole human genome

(~ 5000 clones)

Elimination of non-IBD Fragments

GenomeHIPTM Principle Summary

:Identical By Descent (IBD)regions

KEY:

:Non Identical By Descent (IBD)regions

Identification of all loci implicated in the disease

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Whole genome DNA microarray

12,000 spots in 2 duplicate zones of 24 blocks each

Cy5 : Test DNA

Cy3 : Reference DNA

IntegraChipTM V7: a whole genome BAC-DNA microarray

5244 BAC clonesMedian of 0.5 Mb between clones

Clone identity by end sequencing

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GenomeHIP results• 119 sibpairs (T2D) traited by genomeHIP

• Three most significant linkage results

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Fine-mapping strategy: High density SNP genotyping

SNP selection: Gene centered (+10 kb on each side)

OPA Designer ToolSelection per LD block of the SNP

which presents the best design score(Exclusion of SNPs with score <0.6

or 0.4 for coding SNP)

All SNPs contained in each gene in these 3 regions

Submission to Illumina

Score Design for all SNPs (0 to 1.1)

LD Information from HapMap for each gene,

Using pairwise method, r²=1, maf>0.1 in cauc population

1 OPA containing 1536 tag-SNPs

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Genotyping Production Phase

• Collection– 2068 cases/ controls– 22 plates(96)

• Genotyping Production– 4 plates (96 well format) were genotyped /week (2 FTEs)– 94 samples / plate + 2 CEPH samples (one identical/ plate)

– Allele Calling by clustering

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Key Performances96% of delivered genotypes

• Call Frequency: 92 % of SNPs

threshold >95% of delivered genotypes

• Average Call rate: 96% 97,4% of samples with >95% genotyped SNPs

• Reproducibility: 99.92% Comparison on 1300 SNPs

for 23 times the same sample

Theorical comparison : 29,900

Delivered genotypes: 29,492

Number of discrepancies: 24

(on 21 different SNPs)

• Concordance: 99.45%1328 genotyped SNPs

on 15 samples (CEPH members genotyped on HapMap project)

Theorical comparison : 19,920

Delivered genotypes: 19,502

Number of discrepancies : 107 (around 1/4 on 2 SNPs)

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Relation between Design Score and Call Freq

 With Call freq

<90%With Call freq

>90%

score design

Nber of SNPs

Nber of SNPs %

Nber of SNPs %

0.4 - 0.6 85 18 21,2% 67 78,8%

0.6 - 1 977 51 5,2% 926 94,8%

1.1 474 17 3,6% 457 96,4%

Total 1536 86 5,6% 1450 94,4%

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About IntegraGen www.integragen.com

• IntegraGen is a pioneer in the field of Personalized Healthcare. The Company is dedicated to discovering genes associated with complex diseases and commercializing diagnostic tests to enable individualized diagnosis, prevention and treatment.

• IntegraGen has two commercial operations:– Diagnostic testing and counselling services are offered through

an ISO accredited Diabetes Competency Centre, based in Bonn Germany.

– A Genetic Services business, operating out of the company headquarters in Evry, France, provides customized genotyping and BAC array services to the research community.

• services@integragen.com