Introduction to Affymetrix SNP technology

1. Introduction to Affymetrix SNP technology Jonathan Pevsner, Ph.D. SNP group at the Genome Cafe July 27, 2005

2. Overview of chromosomes and DNA Human genome: 23 pairs of chromosomes 22 autosomal pairs 1 pair of sex chromosomes

3. Scales of DNA Chromosome 10: 135 Mb (983 genes) Chromosome 1: 245 Mb (2580 genes) Human genome: 3 billion base pairs Chromosome 21: 46 Mb (337 genes)

4. Scales of DNA (four bases: A,C,T,G) 1 base pair: may differ (single nucleotide polymorphism, “SNP”) between individuals 800 base pairs: length of typical DNA sequencing read 800 base pairs: convenient size of PCR product 1,000 base pairs: size of a typical protein-coding region 8,500 base pairs: median SNP interval on 100K Affy chip 100,000 base pairs (100 kilobases): size of a typical gene 200,000 base pairs: size of a typical BAC clone 3 million base pairs (3 Mb): smallest size deletion easily visible with conventional cytogenetics 150 Mb: typical chromosome 3,000 Mb (3 Gb): human genome

5. Central dogma of molecular biology protein RNA DNA

6. Across the genome, there are four possible SNP calls: [1] homozygous (AA) [2] homozygous (BB) [3] heterozygous (AB) [4] no call

7. In a deleted region, the possible calls are A, B, or no call. The computer interprets these possible calls as AA, BB, or no call. AA or BB SNP calls are homozygous. Across the genome, there are four possible SNP calls: [1] homozygous (AA) [2] homozygous (BB) [3] heterozygous (AB) [4] no call

9. cell line (control or experimental) hybridize genomic DNA (250 ng) wash digest (XbaI or HindIII) scan ligate adapters acquire raw intensity values PCR amplification normalize fragment and end-label assess SNP intensity values assess SNP LOH values

10. Samples DNA cells Normal female Normal male Affected individual

13. GeneChip DNA Analysis Software (GDAS): .CEL file

16. GeneChip DNA Analysis Software (GDAS) AB call classification .CEL file .CHP file BB AA confidence score dbSNP ID call

17. call classification confidence score “no call”

19. For normal males, there should be three possible SNP calls for the X chromsome: [1] homozygous (A is interpreted by the algorithm as AA) [2] homozygous (B is interpreted by the algorithm as BB) [3] no call Heterozygous (AB) calls should only happen as an error For normal females, there are four possible SNP calls for the X chromosome: [1] homozygous (AA) [2] homozygous (BB) [3] heterozygous (AB) [4] no call

20. 0.100 Probability of a wrong call 0.010 0.001 Called Homozygote n = 7651 n = 52 Called Heterozygote n = 585 No Call 0.001 0.01 0.1 0.25 1 Affymetrix Confidence Score

22. Probe Set: an Affy id Chromosome: 1-22, X, Y, unassigned Physical Position: varies by build! Call: AA, BB, AB, or no call SPA_CN: copy number SPA_pVal: p value for copy number change GSA_pVal: p value for copy number change LOH: -log10 p value for loss of heterozygosity

24. Case #1 SNP data Possible calls: AA, BB, AB, no call Deleted region on chr7: Only expected calls: AA, BB, no call

25. Case #2: SNP arrays - chr 2

26. Case #2: SNP arrays - chr X

27. Web site

28. 700 560 420 count 280 140 consecutive homozygosity size (number of calls)

29. Web site

30. chromosome case childH8 2298 2299 1054 2283 2284 homozygosity stretch 1928 2287 2297 3387 LT MT parent1 parent2

31. chromosome 1 2 3 4 5 6 7 2287 2297 homozygosity stretch 3387

32. 1.4 1.2 1.0 0.8 0.6 0.4 50 40 30 20 10 0 Integration of SNP chip and genomic microarray data A normalized dye ratios B consecutive homozygotes 10 20 30 40 50 physical location, chromosome 22 (megabases) centromere