Introduction to

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Introduction to Affymetrix SNP technology
Jonathan Pevsner, Ph.D. SNP group at the Genome
Cafe July 27, 2005
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Overview of chromosomes and DNA
Human genome 23 pairs of chromosomes 22
autosomal pairs 1 pair of sex chromosomes
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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)
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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
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Central dogma of molecular biology
protein
RNA
DNA
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Across the genome, there are four possible SNP
calls 1 homozygous (AA) 2 homozygous
(BB) 3 heterozygous (AB) 4 no call
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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
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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
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Samples
DNA
cells
Normal female
Normal male
Affected individual
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GeneChip DNA Analysis Software (GDAS) .CEL file
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GeneChip DNA Analysis Software (GDAS)
AB
call classification
.CEL file
.CHP file
AA
BB
confidence score
dbSNP ID
call
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call classification
confidence score
no call
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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
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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
1
0.25
Affymetrix Confidence Score
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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
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Case 1 SNP data
Possible calls AA, BB, AB, no call
Deleted region on chr7 Only expected calls AA,
BB, no call
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Case 2 SNP arrays - chr 2
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Case 2 SNP arrays - chr X
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Web site
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700
560
420
count
280
140
consecutive homozygosity size (number of calls)
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Web site
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chromosome
case
childH8
2298
2299
1054
2283
2284
homozygosity stretch
1928
2287
2297
3387
LT
MT
parent1
parent2
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chromosome
1
2
3
4
5
6
7
2287
2297
homozygosity stretch
3387
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Integration of SNP chip and genomic microarray
data
A
normalized dye ratios
B
consecutive homozygotes
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20
30
40
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physical location, chromosome 22 (megabases)
centromere