Transcript Document

Introduction to
Affymetrix SNP technology
Jonathan Pevsner, Ph.D.
SNP group at the Genome Cafe
July 27, 2005
Overview of chromosomes and DNA
Human genome:
23 pairs of chromosomes
22 autosomal pairs
1 pair of sex chromosomes
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)
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
Central dogma of molecular biology
RNA
DNA
protein
Across the genome, there
are four possible SNP calls:
[1] homozygous (AA)
[2] homozygous (BB)
[3] heterozygous (AB)
[4] no call
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
cell line (control or experimental)
hybridize
genomic DNA (250 ng)
wash
digest (XbaI or HindIII)
scan
ligate adapters
PCR amplification
fragment and end-label
acquire raw intensity values
normalize
assess SNP
intensity values
assess SNP
LOH values
Samples
DNA
cells
Normal
female
Normal
male
Affected
individual
GeneChip DNA Analysis Software (GDAS):
.CEL file
GeneChip DNA Analysis Software (GDAS)
AB
.CEL file
call
classification
.CHP file
BB
dbSNP ID
AA
confidence
score
call
call
classification
confidence
score
“no call”
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
Probability of
a wrong call
0.100
0.010
0.001
Called
Homozygote
Called
Heterozygote
n = 7651
n = 52
n = 585
No Call
0.001
0.01
0.1 0.25
Affymetrix Confidence Score
1
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
Case #1 SNP data
Possible calls:
AA, BB, AB, no call
Deleted region on chr7:
Only expected calls:
AA, BB, no call
Case #2: SNP arrays - chr 2
Case #2: SNP arrays - chr X
Web site
700
count
560
420
280
140
consecutive homozygosity size (number of calls)
Web site
chromosome
case
childH8
homozygosity stretch
2298
2299
1054
2283
2284
1928
2287
2297
3387
LT
MT
parent1
parent2
chromosome
homozygosity stretch
1
2
3
4
5
6
7
2287
2297
3387
consecutive
homozygotes
normalized
dye ratios
Integration of SNP chip and genomic microarray data
A
1.4
1.2
1.0
0.8
0.6
0.4
50
B
40
30
20
10
0
10
centromere
20
30
40
physical location,
chromosome 22 (megabases)
50