Fx Method Development
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Transcript of Fx Method Development
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Genotyping Process Genotyping Process ImprovementsImprovements
- - Focus on Biomek FX Method Development -Focus on Biomek FX Method Development -
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OverviewOverview
Genotyping Assay Prep ReviewGenotyping Assay Prep Review
Process Improvement GoalsProcess Improvement Goals
Process Improvement StrategyProcess Improvement Strategy
Biomek FX Method DevelopmentBiomek FX Method Development
Process ImpactsProcess Impacts
AcknowledgementsAcknowledgements
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Genotyping Assay Prep ReviewGenotyping Assay Prep Review
Nuclease-freeNuclease-freeH2OH2O
2X TaqMan2X TaqManMaster MixMaster Mix
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Genotyping Assay Prep ReviewGenotyping Assay Prep Review
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Genotyping Assay Prep ReviewGenotyping Assay Prep Review
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Process Improvement GoalsProcess Improvement Goals
Why improve a reliable and high-quality process?Why improve a reliable and high-quality process?
ReduceReduce direct cost of consumables and reagents direct cost of consumables and reagents ReduceReduce indirect cost of human resources indirect cost of human resources IncreaseIncrease or maintain genotype throughput or maintain genotype throughput IncreaseIncrease or maintain quality and reliability of data or maintain quality and reliability of data
ReduceReduce our operating cost our operating cost
ReduceReduce our cost per genotype our cost per genotype
IncreaseIncrease competitiveness of Genotyping Services Lab competitiveness of Genotyping Services Lab
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Process Improvement StrategyProcess Improvement Strategy> > R&D EffortsR&D Efforts
NTC Amplification ExperimentNTC Amplification Experiment Towards Towards improvingimproving quality and reliability of data quality and reliability of data
Contamination ExperimentContamination Experiment Towards Towards improvingimproving quality and reliability of data quality and reliability of data
Experimental ProtocolsExperimental Protocols Towards Towards reducingreducing cost of consumables, reagents, cost of consumables, reagents,
and human resourcesand human resources Towards Towards improvingimproving genotype throughput genotype throughput
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Process Improvement StrategyProcess Improvement Strategy> Experimental Pipeline Variables> Experimental Pipeline Variables
1.1. Use Protedyne Biocube to deliver 3uL reaction mixtureUse Protedyne Biocube to deliver 3uL reaction mixtureReductionReduction of reagents of reagents
2.2. Use Biomek FX as sole liquid-handling instrument to deliver 5uL Use Biomek FX as sole liquid-handling instrument to deliver 5uL reaction mixturereaction mixture
ReductionReduction of reagents, consumables, and human resources of reagents, consumables, and human resourcesImprovingImproving genotype throughput genotype throughput
3.3. Use Biomek FX as sole liquid-handling instrument to deliver 3uL Use Biomek FX as sole liquid-handling instrument to deliver 3uL reaction mixturereaction mixture
CombinationCombination of variables 1 and 2 of variables 1 and 2
4.4. Lower annealing temperature in Viper thermocycler protocolsLower annealing temperature in Viper thermocycler protocolsImprovingImproving quality and reliability of data quality and reliability of data
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Process Improvement StrategyProcess Improvement Strategy> R&D Results> R&D Results
Success of Genotyping R&D effortsSuccess of Genotyping R&D efforts Better understanding of NTCsBetter understanding of NTCs Better understanding of how contamination affects our data Better understanding of how contamination affects our data
qualityquality Lowering annealing temperature improves amplificationLowering annealing temperature improves amplification Lower reaction volume of 3 uL Lower reaction volume of 3 uL does notdoes not adversely affect adversely affect
amplification and data qualityamplification and data quality Biomek FXBiomek FX is a viable candidate for consolidating our liquid is a viable candidate for consolidating our liquid
handling needshandling needs
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Biomek FX Method DevelopmentBiomek FX Method Development> > Workflow ExpansionWorkflow Expansion
Experimental Protocols were Experimental Protocols were carried out using the 4SNP/4Q carried out using the 4SNP/4Q workflowworkflow
1SNP/4+Q1SNP/4+QMultiplateMultiplate
8SNP/4Q8SNP/4Q
4SNP/4Q4SNP/4Q
2SNP/4Q2SNP/4Q
1SNP/4Q1SNP/4Q
1SNP/3Q1SNP/3Q
BiomekBiomekFXFX
• Expand Biomek FX Methods Expand Biomek FX Methods to accommodate all of our lab’s to accommodate all of our lab’s workflowsworkflows
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(Biomek FX) Method Development(Biomek FX) Method Development> Guidelines> Guidelines
TimeTime – – streamline individual steps to decrease total runtimestreamline individual steps to decrease total runtime
ConsumablesConsumables – ex. span-8 can replace 96-head / FX tips – ex. span-8 can replace 96-head / FX tips
PrecisionPrecision – how consistent are your results? – how consistent are your results?
AccuracyAccuracy – are you delivering the correct amount of liquid? – are you delivering the correct amount of liquid?
ContaminationContamination – are you contaminating any reagents or products? – are you contaminating any reagents or products?
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Biomek FX Method DevelopmentBiomek FX Method Development> Problems encountered / resolved> Problems encountered / resolved
Improper span-8 positioning to 384-plate wells (stabbing)Improper span-8 positioning to 384-plate wells (stabbing) Framed span-8 using deck position framing tool Framed span-8 using deck position framing tool Adjusted 384-plate labware definition settings Adjusted 384-plate labware definition settings Framed span-8 to specific labware (384-plate) on each deck Framed span-8 to specific labware (384-plate) on each deck
position position
Currently exploring ways to improve liquid deliveryCurrently exploring ways to improve liquid delivery Current system (1mL syringes) can deliver Current system (1mL syringes) can deliver 5uL5uL with c.v. of with c.v. of
+/- 1uL (+/- 1uL (20%20%) upon visual inspection) upon visual inspection
Current system (1mL syringes) can deliver Current system (1mL syringes) can deliver 3uL3uL with c.v. of with c.v. of +/- 2uL (+/- 2uL (67%67%) upon visual inspection) upon visual inspection
Currently exploring advantages and disadvantages of using Currently exploring advantages and disadvantages of using 500 uL and 250 uL syringes, which may improve pipetting 500 uL and 250 uL syringes, which may improve pipetting accuracyaccuracy
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Biomek FX Method DevelopmentBiomek FX Method Development> Implementation> Implementation
Nuclease-freeNuclease-freeH2OH2O
2X TaqMan2X TaqManMaster MixMaster Mix
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Process ImpactsProcess Impacts
Consumables & ReagentsConsumables & Reagents
Eliminate splash lidsEliminate splash lids Eliminate OMM platesEliminate OMM plates Eliminate Axygen (FX) tipsEliminate Axygen (FX) tips Eliminate 24-well bioblockEliminate 24-well bioblock Eliminate Biocube tipsEliminate Biocube tips Eliminate OMM plate reagent dead volumeEliminate OMM plate reagent dead volume Implement 3uL protocols to reduce reaction volume by 40%Implement 3uL protocols to reduce reaction volume by 40%
InstrumentationInstrumentation
Eliminate costly $225,000 / year Protodyne service contractEliminate costly $225,000 / year Protodyne service contract Free up lab space that Biocubes currently occupyFree up lab space that Biocubes currently occupy Allow for greater flexibility and control with regards to internal R&D / Allow for greater flexibility and control with regards to internal R&D /
process improvement / process development effortsprocess improvement / process development efforts
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Process ImpactsProcess ImpactsLIMS / Software ResourcesLIMS / Software Resources Reduce database storage needs by removing one container (OMM)Reduce database storage needs by removing one container (OMM) Reduce software resource time needed to maintain and test the Reduce software resource time needed to maintain and test the
intermediate SOP for new LIMS buildsintermediate SOP for new LIMS builds Reduce scanning and tracking errors by consolidating all liquid transfer Reduce scanning and tracking errors by consolidating all liquid transfer
onto one instrumentonto one instrument
Personnel Resources Reduce personnel resources for processing by 1-2 per pipeline Reduce human plate handling & transport errors by eliminating
intermediate (OMM) container
Additional Benefits Greatly reduce stockroom storage needs Eliminate 100% of recycling and 75% of trash produced by our
lab
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Process ImpactsProcess Impacts> Additional Concerns> Additional Concerns
We lose initial selling point of ‘impressive’ Biocubes with We lose initial selling point of ‘impressive’ Biocubes with potential customers and lab tourspotential customers and lab tours
Need to consider a more robust plate sealing solutionNeed to consider a more robust plate sealing solution
Biomek FX wear and tear
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AcknowledgementsAcknowledgements
David MerrillDavid Merrill
Cinna MonighettiCinna Monighetti
TaqMan Genotyping GroupTaqMan Genotyping Group