High-throughput genotyping

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High-throughput genotyping
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What is genotyping?

• the analysis of DNA-sequence variation
• genotype the genetic constitution of an
  individual

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Alleles

• alternative form of a gene or DNA sequence at a
  specific chromosomal location (locus)
• at each locus an individual possesses two
  alleles, one inherited from the father one from
  the mother
• ? genotype a sum of these two alleles

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Genetic differences

• may cause or predispose to diseases
• determines e.g. individual drug response
• used as markers to identify predisposing genes
  for diseases
• ? high-throughput genotyping technologies needed

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Microsatellite markers

• Di-, tri-, tetranucleotide repeats
• GAACGTACTCACACACACACACATTTGAC
• TTCGATGATAGATAGATAGATAGATACGT

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...Microsatellite markers

• also called
• STR Single Tandem Repeat
• SSR Simple Sequence Repeat
• SSLP Simple Sequence Length Polymorphism
• the number of repeats varies (? 30)
• highly polymorphic
• distributed evenly throughout the genome
• easy to detect by PCR

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SNP markers

• single nucleotide variation
• GTGGACGTGCTTG/CTCGATTTACCTAG

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...SNP markers

• The most simple and common type of polymorphism
• Highly abundant every 1000 bp along human genome
• Most SNPs do not affect on cell function
• some SNPs could predispose people to disease
• influence the individuals response to a drug
• Widely used as genetic markers

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SNP genotyping methods

• over 100 different approaches
• Ideal SNP genotyping platform
• high-throughput capacity
• simple assay design
• robust
• affordable price
• automated genotype calling
• accurate and reliable results

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...SNP genotyping methods

• PCR
• discrimination between alleles
• allele-specific hybridization
• allele-specific primer extension
• allele-specific oligonucleotide ligation
• allele-specific enzymatic cleavage
• detection of the allelic discrimination
• light emitted by the products
• mass
• change in the electrical property

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The Finnish Genome Center

• Independent department of University of Helsinki
• Since 1998
• National core facility for the genetic research
  of multifactorial diseases
• Provides collaboration and genotyping service to
  scientist and research groups in Finland, also
  abroad

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The Finnish Genome Center Goals

• help designing genetic studies
• perform high-throughput genotyping
• perform data analysis
• training of scientists
• adopt and develop new strategies technologies

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Research strategies

• Genome wide scans with microsatellite markers
• 400 dinucleotidemarkers, 10 cM spacing
• Fine mapping
• Project specific (microsatellite) markers
• SNP genotyping
• Primer extension methods (SNuPe and MassArray
  MALDI-TOF), restriction enzyme methods, project
  specific markers

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Genome Scan

• genotyping the whole genome with 400
  microsatellite markers at 10 cM interval
• look for chromosomal regions with excess allele
  sharing

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30
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microsatellite markers at 10 cM distance
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Fine mapping

• candidate regions identified by a genome scan
• candidate genes
• microsatellite or SNP markers
• verification of linkage results

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Setting up PCR-reactions
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Electrophoresis run
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Genotypes
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What the data looks like
Marker Well ID SampleID Allele1 Allele2 Size1 Size
2 D7S513 H01 OA.11616 26 28 190.93 195.02 D7S517 C
07 DYS.5020 26 26 262.19 262.19 D7S640 B02 DYS.381
9 26 29 133.41 139.41 D7S640 G12 OA.1528 26 29 133
.59 139.46 D7S669 E05 OA.11615 26 29 190.37 196.61
D8S258 B06 DYS.5001 26 27 159.38 161.38 D8S260 C0
2 DYS.3931 26 26 215.57 215.57 D8S264 H01 OA.11616
26 26 158.86 158.86
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SNP genotyping at FGC

• PCR-RFLP (restriction fragment length
  polymorphism)
• SNuPe (Single nucleotide primer extension)
• MassARRAY MALDI-TOF (Matrix Assisted Laser
  Desorption/Ionization Time-of-flight mass
  spectrometry)

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PCR-RFLP

• Reactions designed to produce products of
  different sizes after enzymatic cleavage

size in bp
Undigested PCR product
243
C analyte
228
T analyte
94
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SNuPe

• primer extension reactions designed to produce
  differentially labelled products
• analysis by capillary electrophoresis (MegaBACE)

labelled nucleotide
GGACCTGGAGCCCCCACC
Extendable primer
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GGACCTGGAGCCCCCACCC
C analyte
C (blue)
GGACCTGGAGCCCCCACCT
T analyte
T (red)
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MassARRAY System (MALDI-TOF)

• Primer extension reactions designed to generate
  different sized products
• Analysis by mass spectrometry

C/T
G/A
dTTP
dGTP



ddCTP
dATP
G/A



Mass in Daltons
GGACCTGGAGCCCCCACC
Extendable primer
5430.5
GGACCTGGAGCCCCCACCC
C analyte
5703.7
GGACCTGGAGCCCCCACCTC
T analyte
5976,9.9
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Mass spectrometry multiplexing
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Primer extension mass spectrometry

• Advantages
• accurate
• automated assay design
• fast automated data collection
• multiplexing capacity
• Disadvantages
• expensive instruments, consumables
• extensive post-PCR processing

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SNP genotyping workflow at FGC