Jacki BenfieldApplied Markets Division, Applied Biosystems
Validation of HID EVOlution™System: Automation and Integration of Quantification and STR Analysis for High Throughput Sample Processing
© 2008 Applied Biosystems02/23/2008
Validation of HID EVOlution™ System: Automation and Integration of Quantification and STR Analysis for High Throughput Sample Processing
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Disclosure
This presentation will include commercial products from:• Applied Biosystems• Tecan• Other laboratory product suppliers
© 2008 Applied Biosystems02/23/2008
Validation of HID EVOlution™ System: Automation and Integration of Quantification and STR Analysis for High Throughput Sample Processing
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Outline of Presentation
• Scope• Developmental Validation Studies:
• Quantifiler® Human and Y• AmpFℓSTR® STR kits• Complete System Integration
• Summary
© 2008 Applied Biosystems02/23/2008
Validation of HID EVOlution™ System: Automation and Integration of Quantification and STR Analysis for High Throughput Sample Processing
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HID EVOlution™ System: Scope
• Develop validated automated solution to streamline the human identification (HID) workflow
• HID EVOlution™ System• Tecan Freedom EVO® 150
• 4 probe disposable tip configuration• AB custom carriers
• HID EVOlution™ System Software• Designed specifically for AB chemistry and instrumentation• Generates instrument setup files and reports• Seamless transfer of sample information and data
© 2008 Applied Biosystems02/23/2008
Validation of HID EVOlution™ System: Automation and Integration of Quantification and STR Analysis for High Throughput Sample Processing
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Automated Protocols• Quantifiler® Quantification
• Human and Y • Duo (in development)
• 5 dye AmpFℓSTR® PCR Amplification Kits• Identifiler®
• MiniFiler™
• Yfiler®
• 4 dye AmpFℓSTR® PCR Amplification Kits• SGM Plus®
• Profiler Plus®
• COfiler®
• SEfiler™
© 2008 Applied Biosystems02/23/2008
Validation of HID EVOlution™ System: Automation and Integration of Quantification and STR Analysis for High Throughput Sample Processing
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HID EVOlution™ Development and Optimization
• Robot movement • Disposable tip size • Liquid class optimization • Liquid level detection• Mixing protocols• Aspiration/Dispensing mode• Reagent and sample volume requirements• Dilution schemes
Quantifiler® Human and Y Human Male Studies
© 2008 Applied Biosystems02/23/2008
Validation of HID EVOlution™ System: Automation and Integration of Quantification and STR Analysis for High Throughput Sample Processing
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Quantifiler® Kit Validation Studies
• Standard Curve Study• Precision and Accuracy
• Quantification Study• Precision
• 0.025, 0.05, 0.1, 1.0, 5.0 and 25.0 ng/µL of DNA in 12 replicates• Compared to manual operations
• Reproducibility• Precision study - run over three days
© 2008 Applied Biosystems02/23/2008
Validation of HID EVOlution™ System: Automation and Integration of Quantification and STR Analysis for High Throughput Sample Processing
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Quantifiler® Standard Curve Precision and Accuracy Study
Kit Parameter Typical Range Observed Range
Slope -2.9 to -3.3 -3.052 to -3.512
R2 >0.98 0.991 to 0.9999
Slope -3.0 to -3.6 -3.217 to -3.386
R2 >0.98 0.984 to 0.991
Quantifiler® Human (N=75)
Quantifiler® Y (N=40)
© 2008 Applied Biosystems02/23/2008
Validation of HID EVOlution™ System: Automation and Integration of Quantification and STR Analysis for High Throughput Sample Processing
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Quantifiler® Human: Manual vs HID EVOlution™ System
DNA
0.01
0.1
1
10
100
0.025ng/ul 0.05ng/ul 0.1ng/ul 1ng/ul 5ng/ul 25ng/ul
Ave
rage
DN
A Q
uant
ity (n
g/ul
)
ManualPlate1Plate2Plate3Tube1Tube2Tube3
N =12
Sample Normalization and PCR Setup for AmpFℓSTR® Kits
© 2008 Applied Biosystems02/23/2008
Validation of HID EVOlution™ System: Automation and Integration of Quantification and STR Analysis for High Throughput Sample Processing
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DNA Normalization & AmpFℓSTR® Kit Automation Validation
• Accuracy, Precision and Reproducibility - 0.025, 0.1, 0.5, 1.0, 2.0, 5.0,10.0 and 50.0 ng/µL of DNA normalized to target input amount
• Microcentrifuge tube and plate configuration • Each configuration prepared in triplicate• Compared with manual operation• Metrics:
• Genotype concordance• Peak Height• Peak Balance
© 2008 Applied Biosystems02/23/2008
Validation of HID EVOlution™ System: Automation and Integration of Quantification and STR Analysis for High Throughput Sample Processing
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Identifiler®: Precision and Reproducibility Study
Green: Plate
Yellow: Tube
N =171
© 2008 Applied Biosystems02/23/2008
Validation of HID EVOlution™ System: Automation and Integration of Quantification and STR Analysis for High Throughput Sample Processing
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Identifiler®: Manual vs HID EVOlution™ System
Green: Manual
Yellow: Tecan
N =171
0.025 0.1 0.5 2.01.0 5.0 10.0 50.025.00.05 PC
Complete System Integration Study
© 2008 Applied Biosystems02/23/2008
Validation of HID EVOlution™ System: Automation and Integration of Quantification and STR Analysis for High Throughput Sample Processing
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HID EVOlution™ System Validation
Report
SDS Real-time set-up file SDS Real-time
sample results file
CE Instrument set-up File
Report
© 2008 Applied Biosystems02/23/2008
Validation of HID EVOlution™ System: Automation and Integration of Quantification and STR Analysis for High Throughput Sample Processing
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• Automated protocols tested:• Genomic DNA at concentrations: 0.025, 0.1, 0.5, 1, 2, 5, 10,
and 50 ng/µL• 10 replicates per concentration• Quantifiler® Human on a 7500 Real-time PCR System• 4 and 5 dye AmpFℓSTR® Kits • 3130xl Genetic Analyzer
• Position ID (Barcode)• Cross Contamination• Reports
Complete Integrated System Studies
© 2008 Applied Biosystems02/23/2008
Validation of HID EVOlution™ System: Automation and Integration of Quantification and STR Analysis for High Throughput Sample Processing
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Integrated System Validation – Quantification Results
DNA Quantification
0.001
0.01
0.1
1
10
100
CMBi0646
CFBi06
47CMBi06
40CMBi06
38CFB
i0631
CMBi0625
CMBi0628
BMBi0697
CFBi06
21BMBi06
91BFB
i0681
BFBi06
85BFB
i0686
BMBi0671
BMBi0672
BMBi0662
BMBi0669
BMBi0668
CMBi0650
BMBi0655
DN
A Q
uant
ity (n
g/ul
)
Quantifiler® Human quantification results used for normalization
50 510 0.5 0.12 1 0.025
ng/µL
direct1 step2 step
N=10
© 2008 Applied Biosystems02/23/2008
Validation of HID EVOlution™ System: Automation and Integration of Quantification and STR Analysis for High Throughput Sample Processing
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Integrated System Average STR Peak Height Results
AmpFℓSTR® Identifiler® PCR Amplification Kit -10 replicates per DNA concentration
0.025 0.1 0.5 2.01.0 5.0 10.0 50.0 Pos
direct 1 step 2 step
ng/µL N=176
© 2008 Applied Biosystems02/23/2008
Validation of HID EVOlution™ System: Automation and Integration of Quantification and STR Analysis for High Throughput Sample Processing
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Integrated System Heterozygote Peak Height Ratio
N =10
AmpFℓSTR® Identifiler® PCR Amplification Kit
© 2008 Applied Biosystems02/23/2008
Validation of HID EVOlution™ System: Automation and Integration of Quantification and STR Analysis for High Throughput Sample Processing
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Position ID Confirmation Study
• Barcode scanning of sample tubes and tray confirmation• 80 different DNA samples at varying concentrations• The STR genotype for each sample was compared with
the previously generated known genotype
Results• Complete and accurate barcode record• 100% genotype concordance
© 2008 Applied Biosystems02/23/2008
Validation of HID EVOlution™ System: Automation and Integration of Quantification and STR Analysis for High Throughput Sample Processing
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Cross Contamination Study• Samples containing 100ng/uL and TE Buffer only• Two plate configurations• Two runs per configuration
No contamination was observed
Stripe Checkerboard
A
BC
D
E
F
G
H
01 02 03 04 05 06 07 08 09 10 11 12
100 ng/uL
TE BufferABCDEFGH
01 02 03 04 05 06 07 08 09 10 11 12100 ng/uLTE Buffer
© 2008 Applied Biosystems02/23/2008
Validation of HID EVOlution™ System: Automation and Integration of Quantification and STR Analysis for High Throughput Sample Processing
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Quantifiler® Setup Report
© 2008 Applied Biosystems02/23/2008
Validation of HID EVOlution™ System: Automation and Integration of Quantification and STR Analysis for High Throughput Sample Processing
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Quantifiler® PCR Setup Report
© 2008 Applied Biosystems02/23/2008
Validation of HID EVOlution™ System: Automation and Integration of Quantification and STR Analysis for High Throughput Sample Processing
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Normalization/STR PCR Sample Information Report
© 2008 Applied Biosystems02/23/2008
Validation of HID EVOlution™ System: Automation and Integration of Quantification and STR Analysis for High Throughput Sample Processing
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Automation Development and Validation Summary
• Automated protocols and system software have been developed, optimized and validated for the Quantifiler®
Quantification Kits and the AmpFℓSTR® Amplification Kits• Validation studies demonstrated:
• Robust, reproducible and reliable liquid handling • Clean liquid handling capabilities• Accurate data integration and management
© 2008 Applied Biosystems02/23/2008
Validation of HID EVOlution™ System: Automation and Integration of Quantification and STR Analysis for High Throughput Sample Processing
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Advantages of a Turn Key Automated System
• Optimized and validated protocols enable:• Quick implementation• Improved overall first pass success
• Customized software allows for more effective forensic sample processing
• Complete instrument and data integration minimizes:• Assay set-up time• Transcriptional errors • Data review time
For more information visit: evo.appliedbiosystems.com
© 2008 Applied Biosystems02/23/2008
Validation of HID EVOlution™ System: Automation and Integration of Quantification and STR Analysis for High Throughput Sample Processing
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Co-authors and AcknowledgementsR&D
Ada Wong Jason LiuJai ShewaleMike CasselRixun FangRob LagaceSonny YoungVivian T. NguyenManohar Furtado
Technical MarketingJacki Benfield
Product ManagementHeidi Kijenski
TecanDirk AbelnLynda Treat-ClemonsMichael DahmsWendy LauberTony Mamone
© 2008 Applied Biosystems02/23/2008
Validation of HID EVOlution™ System: Automation and Integration of Quantification and STR Analysis for High Throughput Sample Processing
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Legal information
For Research, Forensic or Paternity Use Only. Not for use in diagnostic procedures.
Applera, Applied Biosystems, AB (Design), AmpFLSTR, COfiler, Identifiler, Profiler Plus, SGM Plus, SEfiler, Quantifiler, TaqMan, VIC, and Y-filer are registered trademarks and FAM, MiniFiler, and NED are trademarks of Applera Corporation or its subsidiaries in the US and/or certain other countries.
AmpliTaq Gold is a registered trademark of Roche Molecular Systems, Inc.
EVO® is a trademark of Tecan Corporation.
All other trademarks are the sole property of their respective owners.
HID EVOlution™ System
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