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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 pairs1 pair of sex chromosomes
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Scales of DNA
Chromosome 10: 135 Mb (983 genes)
Chromosome 21: 46 Mb (337 genes)
Chromosome 1: 245 Mb (2580 genes)
Human genome: 3 billion base pairs
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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 read800 base pairs: convenient size of PCR product1,000 base pairs: size of a typical protein-coding region8,500 base pairs: median SNP interval on 100K Affy chip100,000 base pairs (100 kilobases): size of a typical gene200,000 base pairs: size of a typical BAC clone3 million base pairs (3 Mb): smallest
size deletion easily visible with conventional cytogenetics
150 Mb: typical chromosome3,000 Mb (3 Gb): human genome
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proteinRNA
DNA
Central dogma of molecular biology
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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 possiblecalls are A, B, or no call.The computer interprets thesepossible calls as AA, BB, orno 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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genomic DNA (250 ng)
cell line (control or experimental)
digest (XbaI or HindIII)
ligate adapters
PCR amplification
fragment and end-label
hybridize
wash
scan
acquire raw intensity values
normalize
assess SNPintensity values
assess SNPLOH values
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Samples
Normalfemale
Normalmale
Affectedindividual
DNA cells
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GeneChip DNA Analysis Software (GDAS):.CEL file
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GeneChip DNA Analysis Software (GDAS)
.CHP file
.CEL file
AB
AABB
confidencescore
dbSNP IDcall
callclassification
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“no call”
callclassification
confidencescore
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For normal males, there shouldbe three possible SNP calls for the X chromsome: [1] homozygous (A is interpretedby the algorithm as AA)[2] homozygous (B is interpretedby the algorithm as BB)[3] no callHeterozygous (AB) calls shouldonly happen as an error
For normal females, there are four possible SNP callsfor the X chromosome:[1] homozygous (AA)[2] homozygous (BB)[3] heterozygous (AB)[4] no call
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Affymetrix Confidence Score
0.001 0.01 0.1 1
No Call
CalledHeterozygote
CalledHomozygote
0.25
Pro
babi
lity
of
a w
rong
cal
l
n = 7651
n = 52
n = 585
0.001
0.010
0.100
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Probe Set: an Affy idChromosome: 1-22, X, Y, unassignedPhysical Position: varies by build!Call: AA, BB, AB, or no callSPA_CN: copy numberSPA_pVal: p value for copy number changeGSA_pVal: p value for copy number changeLOH: -log10 p value for loss of heterozygosity
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Case #1 SNP data
Deleted region on chr7:Only expected calls: AA, BB, no call
Possible calls: AA, BB, AB, 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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140
280
420
560
700
consecutive homozygosity size (number of calls)
cou
nt
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Web site
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chromosome caseho
moz
ygos
ity s
tret
ch
childH8
2298
2299
1054
2283
2284
1928
LT
MT
parent1
parent2
2287
2297
3387
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2287
2297
3387
1 2 3 4 5 6 7
chromosome
hom
ozyg
osity
str
etch
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A
B
con
secu
tive
hom
ozy
gote
s
physical location, chromosome 22 (megabases)
10 20 30 40 50
nor
ma
lize
d
dye
ra
tios
50
40
30
20
10
0
1.4
1.2
1.0
0.8
0.6
0.4
centromere
Integration of SNP chip and genomic microarray data