1. AFFYMETRIX SNP chips Karin Dahlman-Wright Department of Biosciences and BEA Karolinska Institutet

2. Agenda Introduction Introduction AFFYMETRIX technology AFFYMETRIX technology DNA analysis products DNA analysis products www.affymetrix.com www.affymetrix.com

3. Introduction

4. Milestones in Affymetrix history 1st Microarray Patent Issued 199119921994199519971999 Cover of Science Commercial Launch 2002 IPO 1 st Catalog Product HumanGenome Set (U133) CustomExpress ™ OperateIndependently 1st Expression Product GeneArray ® Scanner 1993 1996 19982000 2001 DNA Analysis 2003 GeneChip ® Scanner 3000 Sacramento manufacturing facility MD Scanner 1990 Affymetrix Japan KK Commercial DNA Microarray prototype Invention of the microarray scanner Invention of the microarray 1989 2004 Human Genome 2.0 Plus array Rat and Mouse Genome 2.0 arrays Nimble Express ™ Photolithography

5. Affymetrix Gene Chips Sequencevariability Expressionvariability

6. Genome analysis Candidate gene analysis Whole genome genotyping CustomSeq™ 30 Kb Mapping 10k and 100k 10,000 and 100,000 genotypes

7. AFFYMETRIX technology; The Gene Chip

8. Photolithography 1 Probe DNA sequences are synthesized in predetermined locations on glass wafers using masks to activate photosensitized DNA with UV light.

9. Photolithography 2 Lamp MaskArray

10. Synthesizing oligonucleotide arrays

11. 49 Chips per Wafer400 Chips per Wafer Manufacturing flexibility

12. 18 µm Millions of copies of a specific oligonucleotide probe >500,000 different complementary probes Single stranded, labeled ‘target’ Oligonucleotide ‘probe’ * * * * * 1.28cm GeneChip Probe Array Hybridized Probe Cell 1.28cm Probe construction

13. 11 µm Millions of copies of a specific oligonucleotide probe >1.300,000 different complementary probes Single stranded, labeled ‘target’ Oligonucleotide ‘probe’ * * * * * 1.28cm GeneChip Probe Array Hybridized Probe Cell 1.28cm Probe construction

14. 8 µm Millions of copies of a specific oligonucleotide probe >2.500,000 different complementary probes Single stranded, labeled ‘target’ Oligonucleotide ‘probe’ * * * * * 1.28cm GeneChip Probe Array Hybridized Probe Cell 1.28cm Probe construction

15. DNA analysis products

16. Affymetrix mapping product timeline 1996 1997 2001 2000 1999 1998 2002 Mapping 10K 2003 HuSNP ® HuSNP ® Mapping Assay: 1,500 SNPs Multiplex PCR assay Allele specific hybridization HuSNP ® Mapping Assay: 1,500 SNPs Multiplex PCR assay Allele specific hybridization

17. GeneChip ® Mapping 10K System NetAffx SNP Annotation GDAS 2.0 Software GeneChip Mapping 10K Arrays GeneChip Mapping 10K Assay GeneChip Scanner 3000

18. Median intermarker distance: 105 kb Mean intermarker distance: 210 kb Mean genetic gap distance: 0.32 cM Average Heterozygosity 0.37 Genome coverage: 11,555 SNPs, Mapping 10 K array

19. Genome coverage; 100K SNP July 2003 NCBI bld 34 Median intermarker distance: 8 kb Mean intermarker distance: 22.5 kb Average Heterozygosity 0.29 Average minor allele frequency 0.21

20. SNP tiling strategy GGTAGCCATGCANGAGTTACTACAG CCATCGGTACGTTCTCAATGATGTC A/G CCATCGGTACGTACTCAATGATGTC PM Allele A MM Allele A CCATCGGTACGTCCTCAATGATGTC CCATCGGTACGTACTCAATGATGTC PM Allele B MM Allele B

21. ”Offset” probes GCCATGCANGAGTTACTACAGTAGC CGGTACGTTCTCAATGATGTCATCG A/G CGGTACGTTCTCTATGATGTCATCG PM + 4 Allele A MM +4 Allele A CGGTACGTCCTCAATGATGTCATCG CGGTACGTCCTCTATGATGTCATCG PM +4 Allele B MM + 4 Allele B

22. Each SNP is interrogated by 40 oligonucleotides 7 quartets were evaluated and 5 were chosen to represent each SNP. Each SNP is interrogated on forward and reverse strand. A total of 10 quartets or 40 probes are used to interrogate one SNP. Dispersed probe pairs. PM A MM B MM A PM B PM A MM B MM A PM B PM A MM B MM A PM B PM A MM B MM A PM B PM A MM B MM A PM B PM A MM B MM A PM B PM A MM B MM A PM B quartet

23. Genomic DNA RE Digestion Adapter Ligation GeneChip ® mapping 10K; Assay overview Xba Fragmentation and Labeling PCR: One Primer Amplification Complexity Reduction AA BB AB Hyb & Wash

24. Assay details 250 ng genomic DNA 50 ng/ul Reduced EDTA Amplify to 250 to 1000 base pair fragments DNA quality control?

25. Array performance   Accurancy: > 99.5 % Concordance with single base extension over 20.000 calls Mendelian inheritance errors: 0.049 % over 30 trios   Reproducibility: 99.96 % 9 samples x 6 replicates 3 caucasians, 3 african american, 3 asian   Call rate: > 90 %

26. GDAS QA report QA report Genotype call Genotype call

27. Discrimination score 1 Ensures there is sufficient specific hybridization PMA MMA PMB MMB d= (PMA-MMA) (PMA+MMA) If MMA>PMA, discard Forward PMA MMA MMB PMB Reverse

28. Discrimination score 2 PMA MMA PMB MMB PMA MMA MMB PMB Calculate 4 median discrimination score Forward Reverse d=Max (Min(dfA, drA), Min(dfB, drB) d fA d > 0.08 Calculate d for SNP

29. RAS score 1 A RAS score is calculated for each quartet R= PMA-MMavg (PMA-MMavg) + (PMB-MMavg) PMA MMA MMB PMB Forward 100 10 R= 100-10 (100-10) + (10-10) = 1.0 PMA MMA MMB Reverse PMB

30. RAS score 2 Determine median RAS score for forward and reverse strand 0 1.0 AA AB BB RASf RASr

31. 10 k SNP array 11 µm * * * * * 18 µm * * * * * 4200 SEK 2050 SEK

32. 100 k SNP array 50 k Xba I 50 k Hind III 8 µm * * * * * 5250 SEK

33. 500 k SNP array 5 µm * * * * * To be launched Q1, 2005 24 probes per SNP

34. Mix and match 10 k 100 k 50 k250 k

35. Affymetrix CustomSeq™ arrays 30kb double-stranded (60 kb total) sequence per chip 30kb double-stranded (60 kb total) sequence per chip sequence can be contiguous or dispersed, any organism sequence can be contiguous or dispersed, any organism Accuracy=>99.99% Accuracy=>99.99% Reproducibility= >99.99% Reproducibility= >99.99% ACGTACGT T T T A T C C T A A C C G T A

36. Resequencing strategy ATCGGTAGCCATGCATGAGTTACTA TAGCCATCGGTACGTACTCAATGAT ATCGGTAGCCATGCATGAGTTACTA TAGCCATCGGTAGGTACTCAATGAT CAGCT GTCGA ATCGGTAGCCATCCATGAGTTACTA TAGCCATCGGTACGTACTCAATGAT ATCGGTAGCCATTCATGAGTTACTA TAGCCATCGGTATGTACTCAATGAT ATCGGTAGCCATACATGAGTTACTA TAGCCATCGGTAAGTACTCAATGAT

37. tiling strategy Resequencing tiling strategy ATCGGTAGCCATGCATGAGTTACTA TAGCCATCGGTACGTACTCAATGAT TCGGTAGCCATGTATGAGTTACTAC AGCCATCGGTAGATACTCAATGATG CAGCT GTCGA TCGGTAGCCATGGATGAGTTACTAC AGCCATCGGTAGCTACTCAATGATG TCGGTAGCCATGCATGAGTTACTAC AGCCATCGGTAGGTACTCAATGATG TCGGTAGCCATGAATGAGTTACTAC AGCCATCGGTAGTTACTCAATGATG

38. Resequencing tiling strategy ATCGGTAGCCATGCATGAGTTACTA TAGCCATCGGTACGTACTCAATGAT CGGTAGCCATGCTTGAGTTACTACA GCCATCGGTACGAACTCAATGATGT CAGCT GTCGA CGGTAGCCATGCGTGAGTTACTACA GCCATCGGTACGCACTCAATGATGT CGGTAGCCATGCCTGAGTTACTACA GCCATCGGTACGGACTCAATGATGT CGGTAGCCATGCATGAGTTACTACA GCCATCGGTACGTACTCAATGATGT

39. Resequencing Tiling Strategy ATCGGTAGCCATGCATGAGTTACTA TAGCCATCGGTACGTACTCAATGAT GGTAGCCATGCATGAGTTACTACAG CCATCGGTACGTACTCAATGATGTC CAGCT GTCGA GGTAGCCATGCAGGAGTTACTACAG CCATCGGTACGTCCTCAATGATGTC GGTAGCCATGCACGAGTTACTACAG CCATCGGTACGTGCTCAATGATGTC GGTAGCCATGCAAGAGTTACTACAG CCATCGGTACGTTCTCAATGATGTC

40. HTA – High throughput assay

41. BEA-NOVUM and Expression Analysis at NOVUM BEA-NOVUM Bio-informatics and Expression Analysis at NOVUM  The objective is to support the KI/KS/HS cluster in the field of gene expression and bio-informatics.  Supported by Karolinska Institute, board of research and Wallenberg Consortium North FunctionsAreas  Provide infrastructureSequence analysis  Provide analysis tools Expression analysis  Provide service Human genetics  Provide education www.bea.ki.se