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. Rousseau IntegraGen SA, www.integragen.com

Strategy Patient collections Bi-polar Disorder Obesity Autism Type 2 Diabetes Others GenomeHIP™ IntegraCHIP tm Access by IntegraGen Access by Partner Objectives: Development of Genetic Tests & Identification of Targets Molecular Diagnostics Target Validation Therapeutic Focus Quicker, Faster, with more precision

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 GenomeHIP TM Principle Summary Identification of all loci implicated in the disease :Identical By Descent (IBD)regions KEY: :Non Identical By Descent (IBD)regions

Whole genome DNA microarray 12,000 spots in 2 duplicate zones of 24 blocks each Cy5 : Test DNA Cy3 : Reference DNA IntegraChip TM V7: a whole genome BAC-DNA microarray 5244 BAC clones Median of 0.5 Mb between clones Clone identity by end sequencing

GenomeHIP results

119 sibpairs (T2D) traited by genomeHIP

Three most significant linkage results

Fine-mapping strategy: High density SNP genotyping SNP selection: Gene centered (+10 kb on each side) OPA Designer Tool Selection 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) LD Information from HapMap for each gene, Using pairwise method, r²=1, maf>0.1 in cauc population 1 OPA containing 1536 tag-SNPs All SNPs contained in each gene in these 3 regions Submission to Illumina Score Design for all SNPs (0 to 1.1)

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

Key Performances Theorical comparison : 19,920 Delivered genotypes: 19,502 Number of discrepancies : 107 (around 1/4 on 2 SNPs) • Concordance: 99.45% 1328 genotyped SNPs on 15 samples (CEPH members genotyped on HapMap project) Theorical comparison : 29,900 Delivered genotypes: 29,492 Number of discrepancies: 24 (on 21 different SNPs) • Reproducibility: 99.92% Comparison on 1300 SNPs for 23 times the same sample 97,4% of samples with >95% genotyped SNPs • Average Call rate: 96% threshold >95% of delivered genotypes • Call Frequency: 92 % of SNPs 96% of delivered genotypes

Relation between Design Score and Call Freq 94,4% 1450 5,6% 86 1536 Total 96,4% 457 3,6% 17 474 1.1 94,8% 926 5,2% 51 977 0.6 - 1 78,8% 67 21,2% 18 85 0.4 - 0.6 % Nber of SNPs % Nber of SNPs Nber of SNPs score design With Call freq >90% With Call freq <90%

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.