validation of hid evolutionâ„¢ system: automation and integration

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

2

Disclosure

This presentation will include commercial products from:• Applied Biosystems• Tecan• Other laboratory product suppliers



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Outline of Presentation

• Scope• Developmental Validation Studies:

• Quantifiler® Human and Y• AmpFℓSTR® STR kits• Complete System Integration

• Summary



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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



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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™



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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



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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



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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)



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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



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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



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Identifiler®: Precision and Reproducibility Study

Green: Plate

Yellow: Tube

N =171



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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



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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



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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



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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



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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



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Integrated System Heterozygote Peak Height Ratio

N =10

AmpFℓSTR® Identifiler® PCR Amplification Kit



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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



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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



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Quantifiler® Setup Report



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Quantifiler® PCR Setup Report



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Normalization/STR PCR Sample Information Report



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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



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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



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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



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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

Thank you

validation of hid evolutionâ„¢ system: automation and integration

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