Sequencing with SNPs and Haplotypes

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Sequencing with SNPs and Haplotypes
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Microarrays Part I and II

• Introduction to DNA and protein microarray
  technology and their applications Mary
• Chromatin immunoprecipitation with microarray
  analysis (ChIP-chip) and data normalization
  Josh
• Next generation sequencing Leslie
• Single nucleotide polymorphism (SNP) chips
  Katie

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Outline of Topics

• What are SNPs and haplotypes?
• How can SNPs be used for genomic sequencing?
• How are SNP-chips designed?
• How can SNP-chip data be used in research?

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Single Nucleotide Polymorphisms (SNPs)

• AGGTCAATG ? AGGCCAATG

SNP alleles must occur in at least 1 of the
population
http//las.perkinelmer.com/content/snps/genotyping
.asp - PerkinElmer Website
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Single Nucleotide Polymorphisms (SNPs)
ggaatttgggtccattcattggatccatgttcagcatttccaaagattaa
aaatgtgaatcataccaatgaaactggcattggctgacttcagaatatca
ccacatcaaacacccagttggcgtcagggaaggaagtttgatgtcactgt
atgttgcctatatgttttcttaacccaacagcaagcttcctgtggaaccc
ctagagctgatggaatgtcttcatagatgacacataggttgtcccaacac
tcctaccatctttttatacttttgagcaaagtcatccagccaaaagccca
tttggaaaataaattctgagttatatacatgagaggacaagagaaaaaga
cttacaaatgacctaAGGcCAATGtcatatagaagatactgagctgggga
aaagctgaaatatgatacatttaccaaaataaatctcacttcgggtcact
attatagttacacggtttaccaatcagccaacttaccatttagatttttt
tcctcttctttttgtcaggagtgttatttacacatgttattcttcttcag
ctaaaactttctctgcttaactaactctggcctatcttgatacttttcca
aaagaaacagagagaaacacactagcagaaagtccaaaggcaccccagga
gtcaaaggcagctttgggaacctttctggactctcaggtctctgagttct
tcagagaccatagaaatcaacaaacgtaggaacaactctattctctagaa
ccaatattttctgtggggaaaaaataaccaaaatttaccaaaagattctg
atagaaactgtcttcatgcttcccctagtggctgggttctcatccagaaa
t
A single SNP occurs among very long stretches of
unchanged DNA.
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Human Genomic SNPs

• Size 3x109 bp
• 99.9 identical between individuals
• SNPs 80-90 of all variation
• 10 million SNPs
• Occur every 100 to 300 bases

http//www.hapmap.org/whatishapmap.html - HapMap
Website
http//www.ornl.gov/sci/techresources/Human_Genome
/faq/faqs1.sht ml - Human Genome Project Website
http//las.perkinelmer.com/content/snps/genotyping
.asp - PerkinElmer Website
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Single Nucleotide Polymorphisms

• Chromosomal markers

• Intragenic Exon-Intron
• Intergenic SNPs promoter, enhancer, non-coding
  RNA, etc

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Intragenic SNPs Exon-Intron
Isoform 1
Isoform 2
jeeves.mmg.uci.edu/hertel/SMAweb/SMAgenetics.html
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Intergenic SNPs
Spleen
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Haplotype
Definition A set of genetic markers on
a chromosome that tend to be inherited together.
SNP 1
SNP 2
gacctaAGG(A,T,G,c)CAATGtcata..tagaagatac(A,T
,G,C)gagctggg
Possible Haplotypes AA, AT, AG, AC, TA, TT, TG,
TC, GA, GT, GG, GC, CA, CT, CG, CC
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Haplotype
Definition A set of genetic markers on
a chromosome that tend to be inherited together.
SNP 1
SNP 2
gacctaAGGcCAATGtcata..
tagaagatactgagctggg
gacctaAGGGCAgctgcatt..
ttggaaagacAgatctgag
Possible Haplotypes AA, AT, AG, AC, TA, TT, TG,
TC, GA, GT, GG, GC, CA, CT, CG, CC
Possible Haplotypes AA, AT, AG, AC, TA, TT, TG,
TC, GA, GT, GG, GC, CA, CT, CG, CC
Possible Haplotypes AA, AT, AG, AC, TA, TT, TG,
TC, GA, GT, GG, GC, CA, CT, CG, CC
99 of Population
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Meiotic Crossovers Events
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Haplotyping Using Tag SNPs
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Haplotyping Using Tag SNPs
Sequence Tag SNPs
Infer sequence of non-tested SNPs
Haplotype
A G
C C
Only need to sequence 300,000 to 600,000 to
deduce the genotype of all 10 million SNPS
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Sequencing with SNPs
PCR
Microarray
G/G A/A A/G
ARMS-PCR
http//www.biomedcentral.com/1471-2164/8/421/
http//www.dddmag.com/uploadedImages/Articles/2008
_01/2008-01-snp-affymetrix1.jpg
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SNP Sequencing by ARMS-PCR
http//nar.oxfordjournals.org/cgi/content/full/29/
17/e88
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SNP-Haplotype Summary

• SNPs AGGTCAATG ? AGGCCAATG
• Haplotype multiple SNPs
• Tag SNPs used to uniquely identify haplotypes
• Meiotic recombination transfer SNPs
• SNP Sequencing PCR low throughput
• Microarray High throughput

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SNP Microarray (SNP-chip) Setup
? Allele (A) and Allele (B)

• 25-mer containing Tag SNP oligonucleotides
• Single basepair mismatches

? MMA and MMB
Allele A
Allele B
.caaaGtccgatc.
. caaaTtccgatc.
Perfect Match Probes (PM)
.aaGtc.
.aaTtc.
Mismatch Probes (MM)
.AaGtc.
.AaTtc.
Anti-sense
.gaCtt.
.gaAtt.
Affymetrix GeneChip Mapping Assay Manual 701441
Rev. 2
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SNP Microarray (SNP-chip) Setup
? Allele (A) and Allele (B)

• 25-mer containing Tag SNP oligonucleotides
• Single basepair mismatches

? MMA and MMB
Affymetrix GeneChip Mapping Assay Manual 701441
Rev. 2
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SNP Microarray (SNP-chip) Setup
? Allele (A) and Allele (B)

• 25-mer containing Tag SNP oligonucleotides
• Single basepair mismatches

? MMA and MMB
A/A A/B
B/B
Affymetrix GeneChip Mapping Assay Manual 701441
Rev. 2
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Sample Preparation
Affymetrix Genome-Wide Human SNP Array 6.0 Data
Sheet
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General Experimental Setup

• SNP-chip sequencing
• Sample set 1 Control
• Sample set 2 Experimental
• Compare SNP profiles Control vs Experimental

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General Experimental Setup



Haplotype Genotype
Phenotype 1
aa, ab, bb
Normal/Diseased 25,000 aa,
ab Diseased bb Normal



500,000 aa, ab, bb Normal/Diseased

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Application of Data

• Population genomics and disease
• Marker assisted breeding
• Individualized medical treatment plans

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Population genomics and disease

• SNP-chip sequencing
• Control population
• Disease population
• Compare SNP profile
• Statistics ? SNP disease association

• Difficulties Control population
• Low frequency alleles
• Analysis

• Single locus vs multi-locus diseases

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Marker Assisted (MA) Breeding
Normal Breeding
MA Breeding
2.5 - 3 yrs
1.5 - 2 yrs

• Use Tag SNPS

• Pick offspring with greatest of genome

• Save ½ to 1½ yr

web.ncifcrf.gov/.../speed_congenics/default.asp
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Further Reading

• SNPs and Haplotyping
• http//www.hapmap.org/
• Syvänen, AC. Nature Reviews Genetics 2, 2001
  930-942
• Meiotic Crossover Events
• Wirtenberger, M. et al. Hum Genet (2005)
  117389-397
• Coop, G. et al. Science (2008) 319 1395-1398
• Nature Reviews (2005) 6 874
• ARMS-PCR
• Ye, et al. Nucleic Acid Research. 2001 (29) Pp
  e88
• SNP-chips
• http//www.affymetrix.com/
• Matsuzaki, H et al. Nature Methods. 2004 (1)
  109 111
• Application in medicine
• Petricoin, et al. Nature Genetics Suppliment.
  2002 (32) 474-479.
• Marker Assisted Breeding
• http//jaxservices.jax.org/speedcongenic.html