A. Holt1, S. Olson1, J. Gabriel, K. Lodhi2, G. Rahi3, J. Adams3, R. Green1 1Human Identification Group, Thermo Fisher Scientific, 180 Oyster Point Blvd., South San Francisco, CA 94080 2Department of Biological Sciences, Fayetteville State University, 1200 Murchison Rd., Fayetteville, NC 28301 3Department of Chemistry and Physics, Fayetteville State University, 1200 Murchison Rd., Fayetteville, NC 28301

ABSTRACT

Recently introduced next generation STR kits are more sensitive, highly robust to inhibitors and highly discriminating. The result of these changes is

that useful STR profiles can now be obtained from previously untypeable forensic DNA samples. Such casework samples often have low quantity

and/or degraded DNA, PCR inhibitors, and, in sexual assault samples, a high quantity of female DNA compared to male DNA. These factors can

make it difficult to decide whether to continue with STR analysis, which STR kit to use and how much DNA to add to the STR amplification reaction.

To address these factors, we have developed a new DNA quantification and assessment kit to provide better correlation between the DNA sample and

resulting STR profile. The Quantifiler® HP and Trio DNA Quantification kits enable efficient and accurate quantification of human DNA and are the first

commercially-available kits to provide a Quality Index to detect the presence of degraded DNA along with PCR inhibitors. In addition, the Quantifiler®

Trio kit determines the quantity of male DNA present in samples. All of these results guide the selection of the most appropriate STR kits in order to

help maximize the chances of casework sample analysis success. These new kits provide a quantitative measure of the degree of DNA degradation,

useful for the determination of how much DNA to add to the STR reaction and which STR kit to use in order to deliver the most informative results.

Through our developmental validation studies we show how degradation predicts the ski slope effect with downstream STR PCR amplification kits and

how the addition of more DNA can recover the lost alleles. We also show how the increase in assay sensitivity and the improved inhibitor tolerance

can be used as a decision making tool to facilitate enhanced efficiency and first pass success rates to obtain complete profiles from challenging

casework samples. These samples include trace DNA samples, highly degraded DNA samples, low quantity of male DNA in high level of female DNA

as well as samples contaminated with PCR inhibitors. The developmental validation data also demonstrates how these new quantification kits provide

critical decision making tools as part of the forensic casework workflow using AmpFLSTR® MiniFiler™, Identifiler® Plus and the GlobalFiler™ Kits.

INTRODUCTION The Quantifiler® Human Plus (“HP”) and Quantifiler® Trio Quantification kits have recently been developed for quantification of human genomic DNA in

forensic samples. As with our previous quantification kits—Quantifiler® Human and Quantifiler® Duo—they use TaqMan® quantitative real-time PCR

technology. However, these next-generation kits have been developed with increased sensitivity and inhibitor tolerance to match next-generation STR

kits. They also include an additional target to assess DNA degradation, and the PCR cycling time has been reduced to 1 hour.

The primary quantification targets (Small Autosomal and Y chromosome) were designed to match the amplicon sizes of typical “mini” STR loci in the

current (“next-generation”) STR kits, for better predictive ability. The Large Autosomal target was designed with a longer amplicon, making it more

susceptible to DNA degradation than the small autosomal target. The ratio of the Small Autosomal to Large Autosomal quantification results

(“Degradation Index”) is a useful predictor of DNA degradation and the resulting ski slope effect in the STR kits. This Degradation Index result is useful

for the determination of which STR kit to use, as well as how much DNA to add to the STR reaction.

MATERIALS AND METHODS

Quantifiler® HP and Quantifiler® Trio kit amplification was performed with 2 µL sample input and 20 µL total reaction volume.

The kits used GeneAmp® 96-well Optical Reaction Plates with clear Optical Adhesive Covers. All data were collected on the

7500 Real-Time PCR system using a 40-cycle amplification protocol that took approximately 1 hour to complete.

Developmental Validation Studies- DNA concentration standards were run on each assay plate; typically 4 to 5 serial dilutions

of the kits’ DNA Standard covering the range of 100 ng/uL to 0.005 ng/uL. Quantification results for Small Autosomal, Large

Autosomal and Y targets were made for each sample, allowing Male:Female ratios to characterize mixture samples, and

Small Autosomal: Large Autosomal ratios to assess the DNA degradation state of samples.

Degraded DNA Studies- The solutions were irradiated with different UV wavelengths for total exposure lengths of 20, 40, 60,

80, 100, or 120 minutes. Samples were not exposed continuously for their designated lengths, but were exposed in intervals

of 20 minutes (See Table 1) with up to 10 seconds lapse between intervals while the lamp was powered off for removal of

exposed samples for selected period of exposure. Each treatment was replicated in triplicate and triplicates were combined

prior to performing experiments.

CONCLUSIONS

Testing with the Quantifiler® HP and Quantifiler® Trio kits demonstrated that the use of multicopy target loci and improved PCR chemistry can significantly improve sensitivity, tolerance to inhibition and overall performance with the most

difficult sample types. The Large Autosomal locus in the multiplex provides information about sample DNA degradation level. Casework labs can use Trio / HP as a valuable decision tool to determine downstream STR reaction DNA input

amounts. Adding more DNA to the STR reaction based on the Degradation Index results may improve the outcome; number of alleles recovered.

TRADEMARKS/LICENSING © 2014 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. For Research, Forensic or Paternity Use Only. Not for use in diagnostic procedure.

Developmental Validation of an Innovative DNA Quantification System

Table 2. Quantifiler® Trio target-dye configuration

The Quantifiler® HP assay is identical in configuration to the Quantifiler® Trio assay, except that it does not contain the

Y chromosome target.

RESULTS PART 1- DEVELOPMENTAL VALIDATION

Quantifiler® HP and Quantifiler® Trio Quantification Kit Detection Sensitivity and Correlation with GlobalFiler Kit Results

The dynamic range of the Quantifiler® Trio assay was tested using serial dilutions of purified human male genomic DNA to obtain concentrations ranging from 120 ng/μL to 0.05 pg/μL in T10E0.1

buffer. These kits were developed to accurately quantify DNA concentrations ranging from 100 ng/uL to 5 pg/µL. For this reason, the listed concentrations for the samples were extrapolated from

Quantifiler HP/Trio kit quantification results for the original stock sample. Quantification assays were performed in parallel with GlobalFiler™ Kit assays for each DNA dilution. (three replicate

reactions with each kit per dilution). For the GlobalFiler™ Kit assay, samples were amplified with 29 PCR cycles on an Applied Biosystems® Veriti® thermal cycler. The STR reactions were analyzed

on an Applied Biosystems® 3500xL Genetic Analyzer. Electropherograms were analyzed with GeneMapper® ID-X Software v1.4. Sample DNA input volumes for Quantifiler® HP and Trio assays

were 2 μL in 20-μL reactions, and for the GlobalFiler™ Kit STR assay, 15 μL (the maximum possible sample volume) in 25-μL reactions. (3 replicate reactions with each kit per dilution). Results for

the DNA dilutions with the highest and lowest DNA concentrations are presented in Table 1.

Sample

Name

Expected

Quantity ng/µL

Quantifiler® Trio DNA Quantification Kit GlobalFiler™ Kit

Average Measured

Quantity of

Small Autosomal Target

(ng/µL)

Average Measured

Quantity of

Y Target (ng/µL)

Average Measured

Quantity of

Large Autosomal Target

(ng/µL)

Average % of Alleles

Recovered (15 uL DNA

input)

Sample 2 100 98.7 86.6 102.6 100

Sample 3 80 83.5 74.0 88.2 100

Sample 4 60 63.1 55.3 67.2 100

Sample 9 1 0.689 0.761 1.001 100

Sample 13 0.03 0.022 0.019 0.025 100

Sample 14 0.01 0.008 0.011 0.012 100

Sample 15 0.00625 0.004 0.004 0.006 88

Sample 16 0.00313 0.002 0.002 0.002 82

Sample 17 0.0016 0.001 0.001 0.001 20

Sample 18 0.0008 0.0006 0.0000 0.0006 4

Sample 19 0.0004 0.0002 0.0002 0.0001 2

Sample 20 0.0002 0.0001 0.0001 0.0001 0

Sample 21 0.0001 0.00005 0.0001 0.0001 0

Sample 22 0.00005 0.0001 0.0002 ---- 0

NTC 0 ---- ---- ---- 0

Locus Name Amplicon

Size (bp)

Chromosome Location(s) Probe Dye/Quencher

Small Autosomal

(SA)

80 Multiple copies on

multiple chromosomes

VIC® dye with MGB quencher

Large Autosomal

(LA)

214 Multiple copies on

multiple chromosomes

ABY® dye with QSY®7

quencher

Y Chromosome (Y) 75 Multiple copies on Y

chromosome

FAM® dye with MGB

quencher

Quantifiler® Trio Quantification Kit Mixture Study

Mixture samples containing 20 pg/μL of human male DNA and increasing amounts of female DNA were prepared. The ratio of male and female DNA in these samples was approximately 1:0,

1:1, 1:5, 1:10, 1:20, 1:100, 1:1000, 1:1,500, 1:2000, 1:4000, and 0:1. The mixture samples were processed for quantification in triplicate using the Quantifiler® Trio DNA Quantification Kit.

Figure 1. Quantification Results for Mixture Study

Figure 1. Quantification Results for Mixture Study. The measured quantification values are approximately equal to the expected values for all ratios tested. The male DNA concentration

stayed consistent across the entire mixture range at approximately 20 pg/μL. For the 1:4000 mixture sample, quantification values measured 84 ng/μL for the SA target which is consistent

with the expected 80 ng/μL value.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

0 5 10 15 20 25 30 35 40 45 50 55

Fra

cti

on

of

All

ele

s R

ec

ove

red

Degradation Index

1 ng

2 ng

3 ng

4 ng

RESULTS PART 2- DEGRADED DNA STUDIES

Quantifiler® Trio Quantification Kit Degradation Index Correlation with STR Results

Human male DNA was isolated from whole human male blood by organic extraction. The DNA was quantified using the Quantifiler® Human kit. The DNA was diluted to 1 ng/µL and exposed to 280, 302 and 365 nm

wavelengths at time intervals of 20 minutes up to 120 minutes. After the degradation step, the DNA was quantified with the Quantifiler® Trio assay and STR analysis was performed using the Ampflstr® Identifiler Plus kit and

GlobalFiler™ kit. As the DNA was degraded for most of the samples, steps were taken to improve recovery of loci. For samples with DI <5, 3 ng DNA was added to the Ampflstr® Identifiler Plus or GlobalFiler™ reaction and for

samples with DI>5, 4 ng DNA was added to the Ampflstr® Identifiler Plus or GlobalFiler™ Reaction. If the DNA concentration was not sufficient to add 3-4 ng DNA, the maximum amount of DNA was added to the reaction.

Figure 5. Ampflstr® Identifiler Plus Results for DNA Sample exposed to 302nm for 100 minutes,

1.8 ng DNA input

FAM Channel

Ski Slope = 3.1

VIC Channel

Ski Slope = 4.7

NED Channel

Ski Slope = 5.1

PET Channel

Ski Slope = 2.7

Figure 2. Ampflstr® Identifiler Plus Results for DNA Sample Exposed to 302nm wavelength for 40

minutes. 1 ng DNA was added to the STR reaction for this sample, the Degradation Index calculated by

the Quantifiler Trio assay is 5.1. The ski slope measure is determined by taking the average peak height

for loci <225 bps/average peak height for loci >225 bps. As shown, the Degradation Index of 5 is

predictive of ski slope pattern in STR reaction.

Figure 2. Ampflstr® Identifiler Plus Results for DNA Sample Exposed to 302nm λ

for 40 minutes, 1 ng DNA input

Figure 4. Ampflstr® Identifiler Plus Results for DNA Sample Exposed to 302nm for 100 minutes,

1 ng DNA input

Figure 4. Ampflstr® Identifiler Plus Results for DNA Sample Exposed to 302nm wavelength for 100 minutes. One ng DNA was added to the STR

reaction for this sample and the Degradation Index calculated by the Quantifiler Trio assay is 35.4. 13 loci (excl. Amelogenin) are recovered with Identifiler Plus

kit when 1 ng DNA is added to the reaction. Triplicate injections were run for this sample, results shown are representative.

Figure 5. Ampflstr® Identifiler Plus Results for DNA Sample Exposed to 302nm wavelength for 100 minutes. Four ng DNA was added to the STR reaction

for this sample and the Degradation Index calculated by the Quantifiler Trio assay is 35.4. 15 loci (excl. Amelogenin) are recovered with Identifiler Plus kitwhen

1.8 ng DNA is added to the reaction and no O/S peaks were obtained for any of the triplcate injections.

Figure 7. Increasing DNA Input of Degraded DNA Samples in Identifiler Plus Reaction

Results in Higher Allele Recovery

Figure 7. Increasing DNA Input of Degraded DNA Samples in Identifiler Plus Reaction Results in Higher Allele Recovery. No O/S peak were

observed with higher input of DNA.

Figure 6. Globalfiler Results for DNA Sample Exposed to 302nm for 100 minutes,

2.7 ng DNA input

Figure 6. GlobalFiler™ Results for DNA Sample exposed to 302nm wavelength for 100 minutes. 2.7 ng DNA was added to the STR

reaction for this sample because the GlobalFiler assay allows for 15 uL input volume, vs. 10 uL input volume with Identifiler Plus kit. 17 loci (excl.

Amel and Yindel) were recovered with GlobalFiler™ assay No O/S peaks were obtained for any of the tripilcate injections.

Degradation Index is Useful for Determining DNA Input and STR Kit Selection

Figure 3. Relationship between Degradation Index & Ski Slope Measure in Identifiler Plus

Figure 3. Relationship between Degradation Index and Ski Slope Measure in

Ampflstr® Identifiler Plus . Only samples with DI </= 50 are shown. Samples with DI>50

are not shown because drop outs were present for one or more dye channels. As shown,

an increase in the DI value results in an increase in the ski slope, with a reasonably linear

relationship obtained up to DI~5-10.

0

5

10

15

20

25

0 10 20 30 40 50

Ski

Slo

pe M

easu

re

DI

FAM

0

1

2

3

4

5

6

7

0 10 20 30 40 50

Ski

Slo

pe M

easu

re

DI

PET

0

2

4

6

8

10

12

14

16

0 10 20 30 40 50

Ski

Slo

pe M

easu

re

DI

NED

0

2

4

6

8

10

12

14

0 10 20 30 40 50

Ski

Slo

pe M

easu

re

DI

VIC Loci <225 bps Loci >225 bps

Sample Name Number of Intervals Interval Length

(min)

Total Time

Irradiated (min) λ_020min 1 20 20

λ_040min 2 20 40

λ_060min 3 20 60

λ_080min 4 20 80

λ_100min 5 20 100

λ_120min 6 20 120

Table 1. Degraded DNA Preparation

Table 1. Degraded DNA Preparation. λ is equal to "365 nm," "302 nm," or "254 nm"

Table 3. Dynamic Range of the Quantifiler® Trio Assay

0.01

0.10

1.00

10.00

100.00

0:1 1:0 1:1 1:5 1:10 1:20 1:100 1:1000 1:1500 1:2000 1:4000

Me

an

Qu

an

t V

alu

e (

ng

/uL

) ,N

=3

Mixture Sample

T. Small Auto Quant

T. Large Auto

T. Y