a comparative cross study of robotic extraction platforms
TRANSCRIPT
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A Comparative Cross Study of Robotic Extraction Platforms for Casework and
Research Applications –Assessing the AutoMate Express™
Heather V. MilnthorpThermo Fisher 2018 Future Trends in Forensic DNA Technology
Foxwoods Resort & Casino – Mashantucket, ConnecticutJune 1st, 2018
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About Our Organization
1970 1994
2010
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Why Choose Automation?
• Kits are already optimized for forensic processing!
• Nucleic acids can be purified from a variety of sample types
• Minimizes sample handling and contamination risks
• Less hands on time for the technician/analyst
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Applied Biosystems®AutoMate Express™ DNA Extraction System
• Compatible kits• PrepFiler Express™• PrepFiler Express BTA™
• N = 13• Recommended incubation time =
40 minutes• Platform run time = < 30 minutes• Elutes in buffer• 7 variable elution volume
options
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PrepFiler LySep™ Column
1.5 mL sample tubeNO LID
LySep™ Column Assembled PrepFiler LySep™ Column
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QIAGEN®EZ1® Advanced XL
• Compatible kits• Many!• Forensic: EZ1 DNA Investigator
Kit • N = 14• Recommended incubation time =
sample type dependent• NMS validated incubation time
= 15 minutes• Platform run time = < 20 minutes• Elutes in buffer or water• 4 variable elution volume
options
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Additional Features
• UV lamp• Direct supply of carrier RNA– Also supplies G2 Buffer and Proteinase K
• Additional protocol options– Trace Protocol– Large Volume Protocol– “Tip Dance” protocol
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QIAGEN®QIAcube® System
• Compatible kits• Most manual kits• Forensic: QIAamp DNA
Investigator Kit• N = 12• Recommended incubation time
depends on protocol / sample type• 1 hour on robot
• Platform run time = > 75 minutes*• Elutes in buffer or water• 20-100µL
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Additional Non-Robotic Extractions Assessed
• PrepFiler™ Forensic DNA Extraction Kit• Organic Extraction
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Outline of “AutoMate” Project
• Brief lysis optimization study for the AutoMate– Length of digest, composition of digest buffer
• Comparative study to other robotic extraction platforms and manual extraction techniques– Sensitivity Study– Case-Type Samples
• Assessment of variable elution volumes for research applications
• Development of differential extraction protocol
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Lysis Optimization for the AutoMate™ Express
• Compare different buffer formulations– PrepFiler™ Lysis Buffer + 1M DTT– QIAGEN® Buffer ATL + Proteinase K– Homebrew Extraction Buffer• TE-4 Buffer, ATL Buffer, 1M DTT, and Proteinase K
• Compare alternate incubation lengths– 40 Minutes– Overnight (16 Hours)
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0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
PFLB-40 ATL-40 EXB-40 ATL-ON EXB-ON
Qua
ntity
of D
NA
Reco
vere
d (n
g/µL
)
Lysis Method
PrepFiler Express™ Lysis Optimization
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Sensitivity
• 20 microliter aliquots of serial blood dilutions– 0.1%– 1%– 10%– 20%– 40%
• 5 replicates of each dilution were extracted per method
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PrepFiler Express™ Kit
0
1
2
3
4
5
6
7
8
9
0.10% 1% 10% 20% 40%
Qua
ntity
of D
NA
Reco
vere
d (n
g/uL
)
Blood Dilution
Automate Express - PrepFiler Kit
0.1% 1% 10% 20% 40%Mean 0.013309 0.159344 1.772271 3.670972 7.016315
Std. Dev 0.003543 0.049541 0.16018 0.22359 1.521231
0
1
2
3
4
5
6
7
8
0.10% 1% 10% 20% 40%Qua
ntity
of D
NA
Reco
vere
d (n
g/uL
)Blood Dilutioni
Linearity - PrepFiler Kit
Observed Expected
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PrepFiler™ Kit (Manual)
0.1% 1% 10% 20% 40%Mean 0.011193 0.220568 2.863981 6.751921 10.17035
Std. Dev 0.001196 0.10863 0.486991 1.116575 0.96186
0
2
4
6
8
10
12
0.10% 1% 10% 20% 40%
Qua
ntity
of D
NA
Reco
vere
d (n
g/uL
)
Blood Dilutions
PrepFiler Manual Extraction
0
2
4
6
8
10
12
0.10% 1% 10% 20% 40%Qua
ntity
of D
NA
Reco
vere
d (n
g/uL
)
Blood Dilutions
Linearity - PrepFiler Manual Extraction
Observed Expected
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PrepFiler Express BTA™ Kit
0
1
2
3
4
5
6
7
8
9
0.10% 1% 10% 20% 40%
Qua
ntity
of D
NA
(ng/
uL)
Blood Dilution
Automate Express - BTA Kit
0.1% 1% 10% 20% 40%Mean 0.005653 0.066038 1.203766 2.100319 3.321876
Std. Dev 0.002619 0.011934 0.277554 0.456226 1.357928
0
0.5
1
1.5
2
2.5
3
3.5
0.10% 1% 10% 20% 40%Qua
ntity
of D
NA
Reco
vere
d (n
g/uL
)
Blood Dilution
Linearity - BTA Kit
Observed Expected
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EZ1® DNA Investigator Kit
0
1
2
3
4
5
6
7
8
9
0.10% 1% 10% 20% 40%
Qua
ntity
of D
NA
Reco
vere
d (n
g/uL
)
Blood Dilutions
EZ1 Investigator Kit
0.1% 1% 10% 20% 40%Mean 0.018249 0.207581 1.780472 3.272231 6.655823
Std. Dev 0.007695 0.027235 0.121364 0.474615 0.654902
0
1
2
3
4
5
6
7
0.10% 1% 10% 20% 40%
Qua
ntity
of D
NA
Reco
vere
d (n
g/uL
)Blood Dilutions
Linearity - EZ1 Investigator Kit
Observed Expected
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0
1
2
3
4
5
6
7
8
9
0.10% 1% 10% 20% 40%
Qua
ntity
of D
NA
Reco
vere
d (n
g/ul
)
Blood Dilution
QIACube - DNA Investigator Kit
QIAamp® DNA Investigator Kit
0.1% 1% 10% 20% 40%
Mean 0.016872 0.140092 1.200939 2.022246 3.08273
Std. Dev 0.003748 0.019389 0.200507 0.237252 0.333108
0
0.5
1
1.5
2
2.5
3
3.5
0.10% 1% 10% 20% 40%Qua
ntity
of D
NA
Reco
vere
d (m
g/uL
)Blood Dilution
Linearity - QIACube
Observed Expected
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Manual Organic Extraction
0
1
2
3
4
5
6
7
8
9
0.10% 1% 10% 20% 40%
Qua
ntity
of D
NA
Reco
vere
d (n
g/uL
)
Blood Dilutions
Organic Extraction (PCIA)
0.1% 1% 10% 20% 40%Mean 0.008494 0.068299 1.128937 2.353379 4.973985
Std. Dev 0.003786 0.007713 0.20709 0.42894 0.69275
0
1
2
3
4
5
6
0.10% 1% 10% 20% 40%
Qua
ntity
of D
NA
Reco
vere
d (n
g/uL
)
Blood Dilutions
Linearity - Organic Extraction
Observed Expected
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Single Factor ANOVA
FS = 12.8183FC = 2.57874P = 4.8E-07
SIGNIFICANT DIFFERENCE
Post-Hoc Tukey Test
QIA >> BTA, ORG (p=0.01)PF > BTA (p=0.05)EZ1 >> BTA, ORG (p=0.01)
0
0.005
0.01
0.015
0.02
0.025
QIA PF BTA ORG EZ1 PFM
Qua
ntity
of D
NA
Reco
verd
(ng/
uL)
Extraction Method
Performance with a 0.1% Blood Dilution
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Single Factor ANOVA
FS = 49.2357FC = 2.57874P = 7.1E-16
SIGNIFICANT DIFFERENCE
Post-Hoc Tukey Test
QIA >> BTA, ORG (p=0.01)PF >> BTA, ORG (p=0.01)EZ1 >> QIA, BTA, ORG (p=0.01)
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
QIA PF BTA ORG EZ1 PFM
Qua
ntity
of D
NA
Reco
vere
d (n
g/uL
)
Extraction Method
Performance with a 1% Blood Dilution
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Single Factor ANOVA
FS = 26.0263FC = 2.58884P = 5.3E-11
SIGNIFICANT DIFFERENCE
Post-Hoc Tukey Test
PF >> QIA, BTA, ORG (p=0.01)EZ1 >> QIA, BTA, ORG (p=0.01)
0
0.5
1
1.5
2
2.5
3
3.5
4
QIA PF BTA ORG EZ1 PFM
Qua
ntity
of D
NA
Reco
vere
d (n
g/uL
)
Extraction Method
Performance with a 10% Blood Dilution
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Single Factor ANOVA
FS = 37.8738FC = 2.58884P = 1.4E-13
SIGNIFICANT DIFFERENCE
Post-Hoc Tukey Test
PF >> QIA, BTA, ORG (p=0.01)EZ1 >> QIA, BTA, ORG (p=0.01)
0
1
2
3
4
5
6
7
8
9
QIA PF BTA ORG EZ1 PFM
Qua
ntity
of D
NA
Reco
vere
d (n
g/uL
)
Extraction Method
Performance with a 20% Blood Dilution
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Single Factor ANOVA
FS = 32.2855FC = 2.58884P = 1.9E-12
SIGNIFICANT DIFFERENCE
Post-Hoc Tukey Test
PF >> QIA, BTA, ORG (p=0.01)ORG >> QIA, BTA (p=0.01)EZ1 >> QIA, BTA, ORG (p=0.01)
0
2
4
6
8
10
12
QIA PF BTA ORG EZ1 PFM
Qua
ntity
of D
NA
Reco
vere
d (n
g/uL
)
Extraction Method
Performance wtih a 40% Blood Dilution
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Sensitivity Study: Take Home Message
• For blood samples…– PrepFiler Express™ >>> PrepFiler Express BTA™– PrepFiler Express™ and EZ1® DNA Investigator Kit
>>> Organic Extraction and QIAamp DNA Investigator Kit
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Case-Type Samples• Touch samples
– 10 per method; all external firearms swabbings• Low level saliva on cotton swatch• Semen/Vaginal fluid mixture• Low level semen on fabric• Low level semen + lubricant on cotton swab• Blood on denim• Blood + humic acid solution• Blood + bleach• Hair with root• Cigarettes
– Unsmoked with saliva placed on to the filter– Smoked
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0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
0.18
0.2
PF BTA OE QIA EZ1 INVEST PFM
Qua
ntity
of D
NA
Reco
vere
d (n
g/µL
)
Extraction Method
Unsmoked Cigarette Butt + 20µL saliva
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0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
PF BTA PFM OE EZ1 QIA
Qua
ntity
of D
NA
Reco
vere
d (n
g/µL
)
Extraction Method
Cutting From a Smoked Cigarette Butt
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0
0.01
0.02
0.03
0.04
0.05
0.06
PF BTA OE QIA EZ1 INVEST PFM
Qua
ntity
of D
NA
Reco
vere
d (n
g/µL
)
Extraction Method
Low Level Semen on Cotton Swab (100 µL of 1:2000 dilution)
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0
0.5
1
1.5
2
2.5
PF BTA OE QIA EZ1 INVEST PFM
Qua
ntity
of D
NA
Reco
vere
d (n
g/µL
)
Extraction Method
5 µL neat blood + 1 µL 2ng/mL humic acid solution
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0
50
100
150
200
250
300
350
PF BTA OE QIA EZ1 INVEST PFM
Qua
ntity
of D
NA
Reco
vere
d (n
g/µL
)
Extraction Method
5 mL of neat semen on a vaginal swab
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0
0.05
0.1
0.15
0.2
0.25
PF BTA PFM OE EZ1 QIA
Qua
ntity
of D
NA
Reco
vere
d (n
g/µL
)
Extraction Method
Low Level Semen with Lubricant
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Platform Highlights
AutoMate™ Express EZ1® Advanced XL
Samples 13 14
Maximum Lysate Volume 500 200/500
Incubation Time 40 minutes 15+ minutes
Run Time 30 minutes 20 minutes
Elution Media Buffer Buffer / H2O
Elution Options 7 4
Instrument List Price ~$44,000 ~$46,000
Kit Size 52 48
Kit List Price $504 / $548 $498
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Variable Elution Volumes
• Interested in assessing the variable elution volumes for research applications– In-House optimized protocols for challenging
sample types utilizes a consumptive dry-down of the extract• Will we get more total DNA with a higher elution
volume?
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0
0.05
0.1
0.15
0.2
0.25
0.3
20 30 40 50 100 200 250Aver
age
Qua
ntity
of D
NA
Reco
vere
d (n
g/µL
)
Elution Volume (µL)
Assessment of Variable Elution Volumes
5.5554 ng total
7.3019 ng total
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Differential Extraction Protocol
• Examine effects of…– Increasing number of sperm pellet wash steps– Single lysis vs. double lysis– Feasibility of using LySep™ Columns for initial
digest
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Comparison of Lysis Workflows
DE1 Lysis 1 - 40 min full speed 5 min remove EF resuspend w/ 50 UPH2O and transfer 1 wash Lysis 3
DE2 Lysis 1 - 40 min full speed 5 min remove EF resuspend w/ 50 UPH2O and transfer 2 wash Lysis 3
DE3 Lysis 1 - 40 min full speed 5 min remove EF resuspend w/ 50 UPH2O and transfer 3 wash Lysis 3
DE4 Lysis 1 - 30 min full speed 5 min remove EF resuspend w/ 50 UPH2O and transfer Lysis 2 1 wash Lysis 3
DE5 Lysis 1 - 30 min full speed 5 min remove EF resuspend w/ 50 UPH2O and transfer Lysis 2 2 wash Lysis 3
DE6 Lysis 1 - 30 min full speed 5 min remove EF resuspend w/ 50 UPH2O and transfer Lysis 2 3 wash Lysis 3
Lysis 1 Lysis 2 Lysis 3490 EXB 390 EXB 470 PFLB10 ProK 10 proteinase K 20 DTT56 deg C 56 deg C 10 proteinase K900 RPM 900 RPM 70 deg C40 or 30 minutes 15 minutes 750 RPM
40 minutes
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To Be Continued…
• Continued development of in-house differential extraction procedure for use with the AutoMate Express™
• Further assessment of extreme variable elution volumes for research applications
• Full validation of purchased instrument– Sensitivity study, reproducibility and precision
study, accuracy and concordance study, contamination assessment, mixture study, mock case samples
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The Center for Forensic Science Research and Education• Heather E. McKiernan• Yih-Ling Saw
NMS Labs• Dr. Heather Degnan• Megan Foley
Arcadia University• Dr. Shanan Tobe
Thermo Fisher Scientific• Kim Wheeler• Megan Meyer• Bernie Scott
Acknowledgements