SequenomSequenom®® MassARRAYMassARRAY®®

DNA DNA MethylationMethylation AnalysisAnalysis

Langdon SmytheLangdon SmytheGenome Analysis Core FacilityGenome Analysis Core Facility

Users Group Meeting Users Group Meeting March 2010March 2010

OverviewOverview

HighHigh--throughput throughput methylationmethylationanalysisanalysis

1. 1. BisulfiteBisulfite treatmenttreatment2. PCR and SAP treatment2. PCR and SAP treatment3. RNA transcription and 3. RNA transcription and cleavage (cleavage (MassCleaveMassCleave™™))4. MADLI4. MADLI--TOF mass TOF mass spectrometer analysisspectrometer analysis

Determines Determines methylationmethylationratios only for covered ratios only for covered ““CpGCpGunitsunits””, not , not CpGCpG sitessites

Past and Current ProjectsPast and Current Projects

Participating labs: 5Participating labs: 5Optimizations: >30Optimizations: >30AmpliconsAmplicons: >70: >70Reactions: ~10,000Reactions: ~10,000Samples: human & mouse, FFPE, breast cancer, Samples: human & mouse, FFPE, breast cancer, laser capture (upcoming)laser capture (upcoming)

PCR Primer DesignPCR Primer Design

User always responsible User always responsible for PCR primer designfor PCR primer designEpiDesignerEpiDesigner Software Software available online available online (http://(http://www.epidesigner.comwww.epidesigner.com/)/)

Core and Core and SequenomSequenom®®

supportsupportPrePre--designed designed EpiPanelEpiPanelavailableavailable

Design ConsiderationsDesign Considerations

Multiple Multiple ampliconsamplicons required to cover a single gene required to cover a single gene ““CpGCpG unitsunits”” contain contain ≥≥1 1 CpGCpG sitesiteMethylationMethylation ratios obtained for ratios obtained for CpGCpG unitsunits

Options forOptions forBisulfiteBisulfite Treatment and PCRTreatment and PCR

1.1. User performs User performs bisulfitebisulfite treatment, PCR treatment, PCR optimization, and PCRoptimization, and PCR

Provide 5Provide 5µµl PCR product in approved 384l PCR product in approved 384--well platewell plateProvide properly formatted Provide properly formatted ampliconamplicon sequences and sequences and plate layoutsplate layouts

Optimization of each Optimization of each ampliconamplicon is essential to the is essential to the success of your projectsuccess of your project

Options forOptions forBisulfiteBisulfite Treatment and PCRTreatment and PCR

2.2. Core performs Core performs bisulfitebisulfite treatment, PCR treatment, PCR optimization, and PCRoptimization, and PCR

Provide sufficient DNA for 500ngProvide sufficient DNA for 500ng--1000ng/sample, 1000ng/sample, ≥≥11ng/11ng/µµl l Provide Provide oligosoligos and properly formatted and properly formatted ampliconampliconsequencessequencesProvide DNA for Provide DNA for ≥≥ 3 rounds of optimization per 3 rounds of optimization per ampliconamplicon

MethylationMethylation AnalysisAnalysis

Core checks data to ensure quality and reports concernsCore checks data to ensure quality and reports concernsAccess to Access to EpiTyperEpiTyper®® software and raw data availablesoftware and raw data available

MethylationMethylation AnalysisAnalysis

MethylationMethylation ratios ratios provided for each provided for each ““CpGCpGUnitUnit”” in Excel formatin Excel format

““EpiGramEpiGram”” provided provided for each for each ampliconamplicon

Time FrameTime Frame

Depends on # of samples and amplicons; post-

optimization & validation

Bisulfite treatment through analysis

4-6 days

2-4

Core performs PCR

-------SAP through data analysisIncludes

Depends on # of amplicons and design; re-design may be necessary

Any # of samples and amplicons; must provide in

approved 384-well plateRestrictions

3 days to > 2 weeks2 daysTime frame

-------2-4# of 384-well plates

Optimization and Validation

User performs PCR

Assumes Core is expecting plates, plates arrive by morning, inpuAssumes Core is expecting plates, plates arrive by morning, input files provided, and no unexpected challengest files provided, and no unexpected challengesPlates containing less than 384 reactions will not be processed Plates containing less than 384 reactions will not be processed fasterfaster

PricingPricing

$70-100$7.70$1500Full Service, 192 rxns

$44-65$5.13$1000Post-PCR, 192 rxns

$30-44 (depends on # of samples)

$3.60$1450Post-PCR, 384 rxns

$43-67 (depends on # of samples)

$5.26$2020Full Service, 384 rxns

Per SamplePer ReactionPer Chip

(Full or half)

Each reaction includes: 1 sample, 1 Each reaction includes: 1 sample, 1 ampliconamplicon, numerous , numerous CpGCpG units and sitesunits and sitesOptimize cost by running 384 reactions at a timeOptimize cost by running 384 reactions at a timeMust consider optimization costMust consider optimization cost

Ex. Upcoming 16 Ex. Upcoming 16 ampliconamplicon optimization and validation estimated at around ~2000optimization and validation estimated at around ~2000

*All prices are approximate

Thank You!Thank You!