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Genome Core Fall Users Group MeetingOct. 15, 2008
• Requesting services, forms, pricing, and sample submission.Kathryn Thompson and Sonia Mirza
• Ambion TaqMan Gene Expression Cells-to-CT Kit: Evaluation ofgene expression data obtained from cell lysates.Langdon Smythe
• SABiosciences RT2 qPCR Arrays: Evaluation of a plate-basedhigh throughput qPCR product. Jennifer Dang
• Sequenom MassArray System: Current successes andchallenges. Kirsten Copren
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The Sequenom MassArray System:Current Successes and Challenges
Kirsten A. Copren, Ph.D.Genome Analysis Core
Oct. 15, 2008
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Sequenom MassArray System
NIH SIG Grant April 2007
1st Project Feb. 2008
Regular operation July 2008
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MassArray Science, the CliffsNotes
1. Single Base PrimerExtension (iPlex-SNPs)
2. Base Specific Cleavage(MassCleave-Methylation)
http://sequenom.com
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http://mysequenom.com
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SNP Workflow
• Assay Design - 1 day• Order Oligos - 1 week• Mix/Adjust Oligos (1-2 days)• Sample preparation (size dependent)• Validation - 3 days• Run: PCR,SAP,iPlex,MS- (4-5 chips/week)• Analysis, Clustering - Quick• Analysis, Verification by Eye - 1 day
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Example Turnaround Times
1. One week (Design, Order, Run):17 SNPs, 1-Plex, 164 samples, 1 Chip
2. Three weeks (Run & Analysis Only):43 SNPs, 2-Plex, 1248 samples, 7 Chips
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Date Species # SNPs Plex # Samples Call Rate StatusFeb-08 Human 25 2 192 99% CompletedMar-08 Mouse 11 1 192 98% CompletedAug-08 Mouse 17 1 164 97% CompletedAug-08 Canine 101 3 102 97% CompletedSep-08 Human 43 2 1248 95-97% CompletedSep-08 Mouse 44 2 ~300 In ProgressOct-08 Human 122 4 ~800 In QueueNov-08 Human 150 4/5 ~1000 DesigningNov-08 Mouse 22 2 80 Designing
SNP Projects
DNA 260/280 > 1.7 recommended
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Project Design Considerations
• Think 384!
• Choose lowest plex level and maximize SNPs perplex.
• Start with more SNPs than desired (~15%)
• 97% Call Rates for successful assays to date
• Validation not necessary. Saves Time.
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Challenges
• System complicated, extensive training andeducation required
• No Pre-PCR liquid handling capacity
• No high throughput DNA quantificationmethod at Cancer Ctr
• Standardize project/sample parameters
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Currently….
• 3/5 of Core Staff trained for independentusage
• Evaluation of Pre-PCR liquid handlerunderway
• Grant submitted for 8-Channel Nanodrop,Pico Green DNA quantification protocol underinvestigation
• Sample/Project submission standardizationunderway
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Methylation Workflow
• Design primers• Optimize PCR (1-4 weeks)• Bisulfite Treat DNA (1 day)• PCR, Run Gel (1/2 day)• Run: SAP,MassCleave, MS (4-5 chips/week)• Data Analysis (Quick)
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Date Species # Amplicons # Samples Total Rxns StatusAug-12 Human 4 96 384 CompletedAug-12 Human 4 45 384 CompletedOct-12 Human 2 20-40 384 Re-Design
Methylation Projects
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Considerations
• PCR primer design and optimization crucial.
• Reaction unit is per amplicon. Range 100-600 bp,300-400 optimal. One gene may require multipleamplicons.
• Bisulfite treatment largest source of unavoidablevariation. Requires 500 ng DNA/sample
• Some success with FFPE with small amplicons (100-150bp)
• Requires analysis of PCR bands on gel to ensuresuccess of run.
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Current Application Development
• Detecting HPV-16 and HPV-18 in samples atattomolar level (<10 copies)
• Fine mapping of chromosomal deletions fromarray CGH data (CNVs)
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Successes
• Excellent SNP call rates
• Methylation analysissuccessful with carefulconsideration to PCR
• “Flexible” system, butoptimal designs most cost-effective
• Quantification applicationsavailable more sensitive thanQPCR method
• Increasing throughputdemands
• Training, education, anddocumentation to increaseunderstanding of workflowand design considerations
• Time required to developand optimize applications
Challenges
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Next Seminar Hosted by Genome Core
Friday, Nov. 7, 2008Sponsored by Sigma Aldrich12:00 – 1:00 p.m., Lurie Conference Room
qPCR Principles and Applications: Assay optimizationtechniques to improve target quantification
Tania Nolan, Ph.D., Global Manager, Sigma-Genosys
Topics:• Improving assay design• Potential issues with template variability• Improving data normalization• Consistency in data analysis
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“The patience to wait until the mud settles and the water runsclear” (Lao Tzu)
USERS
• Elliot Sherr• Nathan Osbun• Rosemary Akhurst• Michael Benizou• Minh Thu Luu• Steve Hamilton• Jennifer Yokoyama• Joe Costello• Chibo Hong• Katie Ridd• Maria DaCosta• Joel Palefsky
SUPPORT & ASSISTANCE
• NIH• Cancer Center• Allan Balmain, Director• Genome Core Staff (Langdon,
Jenny, Kate, Sonia)• Randy Davis• Greg Hamilton• Sequenom: esp. Jodee Steinberg,
Charles Tetzlaff, Pam Shaw