Validation of the Quantifiler Trio and HP DNA Quantification Kits

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

Questions?

kpfoser@nircl.org