SNP Genotyping at HPCGG

1
SNP Genotyping at HPCGG
Alison Brown Lab Manager and Technical
Director Sequenom and Illumina Genotyping Harvard
Partners Center for Genomics and Genetics 65
Landsdowne Street Cambridge, MA
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Sequenom hME Genotyping
Multimek
Bruker Biflex MALDI-TOF
Robodesign spotter
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Sequenom hME Genotyping
Homogeneous Mass Extend (hME) reaction
96ºC, Then 60 ºC
Reagents PCR product, Oligonucleotide Probe,
dNTP/ddNTPs, Thermosequenase, Buffers
ddCTP
dATP
A
C
C
CGGGATAAATATCGACTC
TCACGATGCCCTATTTATAGCTGAGGGGTG
TCACGATGCCCTATTTATAGCTGAGGGGTG
OR
CGGGATAAATATCGACTC
C
A
TCACGAGGCCCTATTTATAGCTGAGTGGTGG
TCACGAGGCCCTATTTATAGCTGAGTGGTGG
G
allele product 5780.8 Da
C
CGGGATAAATATCGACTC
CGGGATAAATATCGACTC
C
A
T
allele product 6094.0 Da
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2001-05 Sequenom Genotyping
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Our requirements in 2005

• Improve/supplement/replace current system
• Higher throughput
• Higher accuracy
• Higher completion rates
• MUCH lower per genotype cost
• Automation of allele calling
• Low use of DNA per reaction

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Choices?

• Sequenom iPLEX
• Existing equipment (or upgrade to Compact MS)
• With existing equipment only 5000 out-lay for
  software upgrade
• Illumina Bead Station
• Already in use by collaborator with excellent
  results
• Require new equipment purchase 260K
• ParAllele
• Require upgrade to existing Affymetrix equipment
• But no spare capacity

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Sequenom iPLEX trial

• Trial carried out in Feb 05
• 121 Sequenom-tested sequences supplied 100
  conversion to assay (4 x 25-plex, 1 x 18-plex and
  1 x 3-plex)
• Two 25-plexes chosen and run on 96 samples
• 45 out of 50 (90 ) of assays yielded genotypes,
  with a success rate of 95.8 with no manual
  editing of calls
• 1 assay showed discordance of 0.78 (3 out of 384
  calls)

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Sequenom iPLEX Genotyping
iPLEX reaction
Reagents PCR product, Oligonucleotide Probe,
mass modified ddNTPs, Thermosequenase, Buffers
ddCTP
ddATP
A
C
C
CGGGATAAATATCGACTC
TCACGATGCCCTATTTATAGCTGAGGGGTG
TCACGATGCCCTATTTATAGCTGAGGGGTG
OR
CGGGATAAATATCGACTC
A
TCACGAGGCCCTATTTATAGCTGAGTGGTGG
TCACGAGGCCCTATTTATAGCTGAGTGGTGG
G
allele product 5754.7 Da
C
CGGGATAAATATCGACTC
CGGGATAAATATCGACTC
A
T
allele product 5778.8 Da
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25-plex iPLEX assay
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Sequenom hME versus iPLEX
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A Gap in the Facility
Sequenom hME
1.50
1.00
Taqman
Sequenom iPLEX
0.50
Illumina BeadStation 384-1536-plex
Affymetrix 100K/500K GeneChip
0.10
10
40
20
30
50
60
400
100K
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Illumina Bead Station

• Training completed
• GoldenGate Genotyping
• Gene Expression/DASL
• Standard operating protocols in place
• Pricing determined
• Automation completed
• DNA quantitation methods
• First project pending May 06

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Small Sample Size Genotyping

• Laboratory of Molecular Medicine
• Currently all diagnostic tests carried out by
  sequencing
• Existing Genotyping Customers
• Repeat individual samples to increase success
• Confirm interesting results in mutation studies

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Existing platform

• Taqman
• Location problems
• Equipment at capacity
• Sequenom hME or iPLEX
• Too high throughput, chip costs make it
  prohibitive
• Manual merging of samples and different tests
  difficult

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Robotics and Informatics

• Robotics support all Core Facilities
• Pre-PCR Biomek FX
• Post-PCR Biomek FX and Multimek
• Require guaranteed daily access to robotics
• Substantial Informatics support required
• LIMS
• Robotic programming

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New Platform

• Luminex/Tm Biosciences
• Existing diagnostic tests, e.g. Cystic Fibrosis
• Custom SNP panels
• Reagents only in wells with samples, no chip
  costs
• To be tested in the next few months
• Illumina/Cyvera Technology
• Microbead assay technology containing holographic
  code
• Expected release end of of 2006
• Little info released yet
• Equipment price 100K
• Minimum 10 samples?
• Per genotype price?
• Alpha and Beta testing performance?

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HPCGG Sequenom and Illumina Genotyping Facility

• Faculty Director Dr David Kwiatkowski, MD PhD.
• Technical Research assistant - Maura Regan
• Services
• Sequenom hME and iPLEX genotyping
• Illumina GoldenGate Genotyping
• Fragment analysis and Spectratyping
• For more information
• www.hpcgg.org/Genotyping