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Author: Ken Pfoser, DNA Technical Leader, Northeastern Illinois Regional Crime Laboratory Quantifiler® Trio Evaluation, Summary: * Reproducible results - Replicated Sample - Replicated Standards * Easy to automate - Less standards to set up * Faster amplification time (60 min) * Degradation Index * M:F Ratio
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Validation of the Life Technologies Quantifiler® Trio & HP DNA Quantification Kits (2014)
Ken Pfoser DNA Technical Leader Northeastern Illinois Regional Crime Laboratory
Northeastern Illinois Regional Crime Laboratory (NIRCL)
Vernon Hills, IL (Lake County)
Full Service Laboratory Biology/DNA/Parentage Drug Chemistry Toxicology Fingerprints/Footwear Firearms/Toolmarks Crime Scene Support
ASCLD-LAB 1997,2002,2007 ISO FQS 2012
NIRCL DNA
(2) 310s – Genetic Analysis (1) 7500 – Quantification (1) Qiagility – Amp and Quant
Setup (2) EZ1 Adv. XL, Investigator
Kit - Extraction Some organic extraction –
sperm (F2), bone, problem touch samples.
900 Biology/DNA Cases (2013) 4 DNA analysts 1 Biology analyst Average turn-around 30-40 days 300-500 CE samples/month Backlog <100
Quantifiler® Trio New Quant Kit
Two Human Targets (Trio and HP) Small Autosomal Target Large Autosomal Target
Male Target (Trio only) IPC New Reporter Dyes
VIC®
ABY™ 6-FAM™ JUN™ Mustang Purple™
Quantifiler® Trio
Quantifiler Trio® Kit Components Primer Mix (4 tubes) PCR Reaction Mix (4 tubes) Dilution Buffer (1 Tube) DNA Standard 100ng/ul (1 tube, separate box)
HID Real Time PCR Analysis Software
• Customize Templates
Quantifiler® Trio
7500 Real Time calibration ROI Background Optical Dye Rnase P Instrument
Verification
Quantifiler® Trio & HP Validation
Qiagility Setup (2013; Trio) Reproducibility (2013; Trio) Precision (2013; Trio) Sensitivity (2013, 2014; Trio/HP) Contamination (2013; Trio) Case Samples (2013, 2014; Trio/HP) Mixtures (2014; Trio/HP) Inhibition (2014; Trio/HP)
Quantifiler® Trio & HP Validation
Qiagility Setup for Quantifiler® Trio (HP same setup)
Reagent Placement Standard Dilutions (20ng, 2ng, 0.2ng, 0.02ng, 0.002ng) [2013]
Standard Dilutions (50ng, 5ng, 0.5ng, 0.05ng, 0.005ng) [2014]
Reagent Configuration (8ul Primer, 10ul Reaction Mix) Sample Configuration (2ul sample) Sample Placement Plate Setup
Quantifiler® Trio Validation
Qiagility Setup
Quantifiler® Trio Validation
Reproducibility (2013) Standard dilution set (X6) IPC 26.8-28.6 Ct Large Autosomal
Slope = -3.3 R2 = 0.99
Small Autosomal Slope = -3.1 R2 = 0.99
Male Slope = -3.1 R2 = 0.99
Quantifiler® Trio Validation Reproducibility (2013)
Standard dilution set (X8)
Neat (100ng) Large: Average 114.7, STD 6.9 Small: Average 151.4, STD 19.8 Y: Average 127.8, STD 9.3
Standard 1 (20ng) Large: Average 22.1, STD 1.8 Small: Average 25.6, STD 5.6 Y: Average 21.9, STD 2.9
Standard 2 (2ng) Large: Average 2.2, STD 0.1 Small: Average 2.3, STD 0.4 Y: Average 2.0, STD 0.1
Quantifiler® Trio Validation Reproducibility (2013)
Standard dilution set (X8)
Standard 3 (0.2ng) Large: Average 0.19, STD 0.01 Small: Average 0.18, STD 0.01 Y: Average 0.17, STD 0.01
Standard 4 (0.02ng) Large: Average 0.02, STD 0.002 Small: Average 0.02, STD 0.01 Y: Average 0.02, STD 0.008
Standard 5 (0.002ng) Large: Average 0.002, STD 0.0007 Small: Average 0.003, STD 0.003 Y: Average 0.001, STD 0.0009
Quantifiler® Trio Validation Reproducibility (2013)
Buccal Swab Sample (X20) Large
Average = 0.2827 STD = 0.03 High = 0.3522, Low = 0.2164
Small Average = 0.2706 STD = 0.05 High = 0.4206, Low = 0.1861
Y Average = 0.1953 STD = 0.02 High = 0.2405, Low = 0.1679
Quantifiler® Trio Validation Sensitivity (2013)
Standard dilution set extended out to Standards 6 & 7 Standard 6 (0.0002)
0.0001 (L); 0.0002 (S); Blank (Y) 0.0002 (L); Blank (S); 0.0001 (Y) 0 (L); 0.0001 (S); 0.0001 (Y) Blank (L); 0.0002 (S); 0.0001 (Y) Blank (L), Blank (S), Blank (Y) 0.0001 (L); 0.0004 (S), Blank (Y)
Standard 7 (0.00002) Blank (L,S,Y) (x3) Blank (L), 0.0002 (S), Blank (Y) 0 (L); 0.0001 (S), Blank (Y) Blank (L); Blank (S), 0.0002 (Y)
Quantifiler® Trio& HP Validation Sensitivity (2014)
Two human genomic DNA samples at 1000pg/ul were diluted to final concentrations of: 1000, 100, 10, 5, 2.5, 1.25, 0.62, 0.31, 0.15 and
0.08 pg/ul and run in triplicate Quanted with Trio and HP Amplified in ID+ to obtain allele counts
Quantifiler® Trio Validation Sensitivity (2014) Quant data Sample 1 (ng/ul) Sample (Y) Obs. (LA) Obs. (SA) Obs. Exp. 1A1 0.9993 1.1218 1.0305 1 1B1 0.0989 0.1137 0.1074 0.1 1C1 0.0107 0.0107 0.0124 0.01 1D1 0.0046 0.0037 0.0042 0.005 1E3 0.0030 0.0023 0.0035 0.0025 1F2 0.00190 0.00110 0.00160 0.00125 1G3 0.00060 0.00140 0.00070 0.00062 1H3 0.00040 0.00030 undet 0.00030 1I2 undet undet undet 0.00015 1J1 undet undet 0.0002 0.00008
Inconsistencies with expected data and replicates at and below 0.0025 (2.5 pg)
Quantifiler® Trio Validation Sensitivity (2014) Quant data Sample 2 (ng/ul) Sample (Y) Obs. (LA) Obs. (SA) Obs. Exp. 2A1 1.2185 1.0667 1.0501 1 2B1 0.1150 0.0899 0.0761 0.1 2C1 0.0138 0.0077 0.0077 0.01 2D1 0.0039 0.0033 0.0021 0.005 2E1 0.0020 0.0020 0.0018 0.0025 2F2 0.00130 0.00050 0.00050 0.00125 2G3 0.00050 0.00030 0.00030 0.00062 2H1 0.00030 0.00020 0.00030 0.00030 2I2 0.00020 0.00010 0.0010 0.00015 2J1 undet undet undet 0.00008
Quantifiler® HP Validation Sensitivity (2014) Quant data Sample 1 (ng/ul) Sample (LA) Obs. (SA) Obs. Exp. 1A1 0.7849 0.7677 1 1B1 0.0727 0.0752 0.1 1C1 0.0172 0.0193 0.01 1D1 0.0029 0.0050 0.005 1E2 0.0013 0.0023 0.0025 1F2 0.00150 0.00120 0.00125 1G1 0.00050 0.00040 0.00062 1H2 0.00030 0.00030 0.00030 1I1 undet 0.0001 0.00015 1J2 undet undet 0.00008
Inconsistencies with expected data and replicates at and below 0.0025 (2.5 pg)
Quantifiler® HP Validation Sensitivity (2014) Quant data Sample 2 (ng/ul) Sample (LA) Obs. (SA) Obs. Exp. 2A1 0.6942 0.7105 1 2B1 0.0631 0.0636 0.1 2C1 0.0031 0.0036 0.01 2D1 0.0023 0.0023 0.005 2E1 0.0013 0.0009 0.0025 2F1 0.00060 0.00020 0.00125 2G1 0.00020 0.00010 0.00062 2H2 0.00010 0.00020 0.00030 2I1 0.00010 0.0010 0.00015 2J1 undet undet 0.00008
Quantifiler® Trio Validation Sensitivity (2014) Sample 1: Allele Counts ID+ on 310 @ 75 rfu AT
0.01 ng/ul = 28/29, 29/29, 28/29 0.005 ng/ul = 22/29, 20/29, 18/29 0.0025 ng/ul = 10/29, 5/29, 11/29 0.00125 ng/ul = 0/29, 0/29, 1/29 0.00062 ng/ul = 0/29, 1/29, 1/29 0.0003 ng/ul = 0/29, 0/29, 0/29 0.00015 ng/ul = 0/29, 0/29. 0/29 0.00008 ng/ul = 0/29, 0/29, 0/29 Cutoff = 0.001 ng/ul
Quantifiler® Trio& HP Validation Sensitivity (2014) Sample 2: Allele Counts ID+ on 310 @ 75 rfu AT
0.01 ng/ul = 9, 1, 8 0.005 ng/ul = 3, 2, 6 0.0025 ng/ul = 3, 3, 0 0.00125 ng/ul = 0, 0, 0 0.00062 ng/ul = 0, 0, 0 0.0003 ng/ul = 0, 0, 0 0.00015 ng/ul = 0, 0, 0 0.00008 ng/ul = 0, 0, 0
Quantifiler® Trio Validation Contamination (2013)
No contamination observed on previous Qiagility validations
Alternating rows of dilution buffer = No DNA Sporadic insertion of dilution buffer wells = No DNA
Quantifiler® Trio Validation Mixture (2014)
6 Mixed samples provided M:F (1:0, 1:1, 1:20, 1:100, 1:1000, 0:1) Run in triplicate Sample 3: 0.0259 (LA), 0.0302 (SA), 0.0238 (Y) Sample 4: 0.0465 (LA), 0.056 (SA), 0.0368 (Y) Sample 5: 0.668 (LA), 0.645 (SA), 0.0413 (Y) Sample 6: 2.3802 (LA), 2.0792 (SA), 0.0334 (Y) Sample 7: 26.9761 (LA), 25.6541 (SA), 0.0377 (Y) Sample 8: 0.0264 (LA), 0.0271 (SA), undet (Y)
Quantifiler® HP Validation Mixture (2014)
Same 6 Mixed samples for Trio M:F (1:0, 1:1, 1:20, 1:100, 1:1000, 0:1) Run in duplicate (ran out of sample) Sample 3: 0.0363 (LA), 0.0368 (SA) Sample 4: 0.0716 (LA), 0.061 (SA) Sample 5: 1.1946 (LA), 1.1305 (SA) Sample 6: 5.3981 (LA), 4.8894 (SA) Sample 7: 31.7595 (LA), 29.2962 (SA) Sample 8: 0.0513 (LA), 0.0441 (SA)
Quantifiler® Trio & HP Validation Inhibition (2014)
Human DNA samples spiked with inhibitors 0.1 ng/ul DNA w/ 200uM Hematin (#9) 0.1 ng/ul DNA w/ 800 uM Hematin (#10) 0.1 ng/ul DNA w/ 200 ng/ul Humic Acid (#11) 0.1 ng/ul DNA w/ 500 ng/ul Humic Acid (#12)
Trio #9: 0.0546 (LA), 0.0521 (SA), 0.0540 (Y) IPC 26.9 Trio #10: undet (LA), 0.0350 (SA), 0.0096 (Y) IPC undet Trio #11: 0.0309 (LA), 0.1182 (SA), 0.0753 (Y) IPC 27.0 Trio #12: undet (LA), 0.1306 (SA), 0.0319 (Y) IPC undet HP #9: 0.0804 (LA), 0.0631 (SA) IPC 27.2 HP #10: undet (LA), 0.0319 (SA) IPC undet HP #11: 0.0684 (LA), 0.1307 (SA) IPC 27.2 HP #12: undet (LA), 0.1333 (SA) IPC undet
Quantifiler® Trio Validation Case Type Samples (2013, 2014)
Variety of case type samples tested Clothing Toothbrush Cigarette butt Touch (gun, steering wheel, tape) Blood samples Buccal swab standard Blood standard
Trio results replicated (2013) & compared to Duo
Degradation Index (DI) Amplified in ID+
Quantifiler® Trio Validation Case Type Samples (2013)
Example 1 (Clothing) Trio
0.2659 (LA) 0.7567 (SA) 0.5181 (Y)
Duo 0.267 (H) 0.258 (M)
DI = 2.846; IPC 25.9 All loci present
Quantifiler® Trio Validation Case Type Samples (2014)
Example 2 (body swab) Trio
2.4033 (LA) 4.4357 (SA) 0.0005 (Y)
Duo 0.3871 (H) 0.0013 (M)
DI = 1.846; IPC 27.5 All alleles present
Quantifiler® Trio Evaluation Case Type Samples (2013)
Example 3 (Touch; gun) Trio
0.0047 (L) 0.026 (S) 0.0177 (Y)
Duo 0.00582 (H) 0.0109 (M)
DI = 5.498; IPC 25.8 9 loci with data
Quantifiler® Trio Evaluation Case Type Samples (2013)
Example 4 (Touch; car) Trio
0.0041 (L) 0.0029 (S) 0.0025 (Y)
Duo 0.00355 (H) undet (M)
DI = 0.706, IPC 26.1 15 loci with data
FGA no data
HID Data
HID Data Export
• Select specific data to export in table format • Easily export into Excel & Word to work with current
worksheets
HID Data
Quantifiler® Trio Evaluation Summary
Reproducible results Replicated Sample Replicated Standards
Easy to automate Less standards to set up
Faster amplification time (60 min) Degradation Index M:F Ratio
Quantifiler® Trio Evaluation Future Considerations
Mixtures Evaluate mixture ratios correlation
with DI and M:F ratio calculations
Stochastic Studies Evaluate DI values and stochastic
effects on amplified samples
Acknowledgements NIRCL Kelly Lawrence Sarah Owen Michelle Thomas Chrissy Weathers
Life Technologies
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