Developmental validation of QIAGEN Investigator® 24plex QS Kit and Investigator® 24plex GO! Kit: Two 6-dye multiplex assays for the extended CODIS core loci

Forensic Science International: Genetics 29 (2017) 9–20

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Forensic Science International: Genetics journal homepage: www.elsevier.com/locate/fsig

Research paper

Developmental validation of QIAGEN Investigator1 24plex QS Kit and Investigator1 24plex GO! Kit: Two 6-dye multiplex assays for the extended CODIS core loci Melanie Kraemera , Anke Prochnowa , Michael Bussmanna , Mario Scherera,* , Ralf Peista , Carolyn Steffenb a b

QIAGEN GmbH, QIAGEN Strasse 1, 40724 Hilden, Germany National Institute of Standards and Technology, Applied Genetics Group, 100 Bureau Drive, Gaithersburg, MD 20899, USA

A R T I C L E I N F O

Article history: Received 4 November 2016 Received in revised form 6 February 2017 Accepted 9 March 2017 Available online 12 March 2017 Keywords: Investigator1 24plex QS Kit Investigator1 24plex GO! Kit Forensic validation studies STR Rapid identification Direct amplification

A B S T R A C T

The original CODIS database based on 13 core STR loci has been overwhelmingly successful for matching suspects with evidence. In order to increase the power of discrimination, reduce the possibility of adventitious matches, and expand global data sharing, the CODIS Core Loci Working Group determined the expansion of the CODIS core loci to 20 STR plus three additional “highly recommended” loci (SE33, DY391, Amelogenin) Hares, 2015, 2012 [1,2]. The QIAGEN Investigator 24plex QS and Investigator 24plex GO! Kits are 6-dye multiplex assays that contain all markers of the expanded 23 CODIS core loci along with a unique internal performance control that is co-amplified with the STR markers. The “Quality Sensor“ generates additional information for quality control and performance checks. Investigator 24plex QS is designed for purified DNA from casework and reference samples, whereas 24plex GO! is dedicated to direct amplification of reference samples, like blood or buccal cells on FTA or swabs. A developmental validation study was performed on both assays. Here, we report the results of this study which followed the recommendations of the European Network of Forensic Science Institutes (ENFSI) [3] and the Revised Validation Guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM) [4]. Data included are for PCR-based procedures e.g. reaction conditions, effects of PCR annealing temperature variations, amplification cycles or cyclers, sensitivity (also in the context of the Quality Sensor), performance with simulated inhibition, stability and efficiency, precision, reproducibility, mixture study, concordance, stutter, species specificity, and case-type samples. The validation results demonstrate that the Investigator 24plex QS and Investigator 24plex GO! Kits are robust and reliable identification assays as required for forensic DNA typing in forensic casework analysis and databasing. © 2017 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NCND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction Based on the CODIS Core Loci Working Group determination to expand the CODIS core loci to 20 STR plus three additional “highly recommended” loci [1,2], QIAGEN has developed the Investigator 24plex Kits. The Investigator 24plex QS Kit is specifically designed for analysing challenging samples of low quality and quantity from casework whereas Investigator 24plex GO! is specifically designed to facilitate the processing of reference samples

* Corresponding author. E-mail address: [email protected] (M. Scherer).

collected for the purpose of database submissions. These 6-dye multiplex assays co-amplify the 22 polymorphic STR markers D1S1656, D2S441, D2S1338, D3S1358, D5S818, D7S820, D8S1179, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433, D21S11, D22S1045, CSF1PO, FGA [FIBRA], TH01 [TC11], TPOX, vWA, SE33 [ACTBP2], and DYS391, the gender-specific Amelogenin and the Quality Sensor. All genetic loci have been characterized in numerous studies by other laboratories as examples: [5– 7]. As a special feature, both kits contain an internal PCR control (Quality Sensor QS1 and QS2), which provides valuable data without affecting PCR performance, even in the case of low DNA content samples. The internal Quality Sensor is enclosed in the Primer Mix and amplified simultaneously with the sample’s DNA

http://dx.doi.org/10.1016/j.fsigen.2017.03.012 1872-4973/© 2017 The Authors. Published by Elsevier Ireland Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-ncnd/4.0/).

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to show if the PCR amplification was successful, to distinguished between failed PCR amplification from absence of DNA, as well as inhibited DNA from degraded DNA. 2. Materials and methods 2.1. DNA and direct amplification substrates Control DNA 9948 was obtained from QIAGEN. Unless otherwise stated, the DNA from anonymous donor samples (blood, saliva, buccal swabs or mocked case-type samples, XX107 used for mixture study) was extracted by using the QIAamp DNA Investigator Kit, EZ1 DNA Investigator Kit or the QIAsymphony DNA Investigator Kit following manufacturer’s instructions and quantitated by QIAGEN Investigator Quantiplex Kit or Investigator Quantiplex HYres Kit on the Rotor-Gene Q (QIAGEN) or the Applied Biosystems 7500 Real-Time PCR System. Tests with the Investigator 24plex GO! Kit were performed with blood on FTA, blood on non-FTA, buccal cells on FTA and buccal swabs. Blood on FTA samples were created by spotting 125 ml of whole blood onto the center of the sampling area on a nonindicating FTA Micro Card (GE Healthcare) or on an EasiCollect Card (GE Healthcare). Blood on non FTA samples were created by spotting 125 ml of whole blood on a 903 Proteinsaver Snap-Apart Card (GE Healthcare). Buccal cells on FTA were created following the instructions for EasiCollect Cards (GE Healthcare) or the Nucleic-Cards (Copan). Buccal swabs were created with the Puritan Sterile Cotton tipped Applicators; Puritan Sterile Polyester tipped Applicators, Sarstedt Forensic Swabs, GE Healthcare Omni Swabs, or Copan Flocked Swabs. Control DNA 9948 was diluted in Nucleic Acid Dilution Buffer (QIAGEN) for sensitivity studies. The sensitivity for the Investigator 24plex GO! Kit was tested using serial dilutions of whole blood spotted onto non-indicating FTA Micro Card (GE Healthcare) or using serial dilutions of swab lysates. The samples for mixtures testing were created by mixing control DNA 9948 and XX107 in ratios of 1:1, 3:1, 7:1, 10:1, 15:1 and vice versa. The species specificity was tested using 2.5 ng of DNA from vertebrate species (cow, pig, horse, sheep, goat, chicken, mouse, rat, hamster, rabbit, cat and dog), or 500 pg of DNA from primates (chimpanzee, bonobo, gorilla, orangutan and macaque). 2.2. Pre-PCR sample preparation for direct amplification For blood or buccal cells on cards a 1.2 mm disc was punched and added to the PCR reaction without any pre-treatment. For buccal cells on treated paper, 2 ml Investigator STR GO! Punch Buffer was added to the reaction. In automated procedures, a STAR Q Punch AS instrument was used for card handling, punching and PCR setup. Buccal cells on swabs were lysed in 500 ml STR GO! Lysis Buffer at 95  C for 5 min shaking at 1200 rpm in a thermomixer. 2 ml of swab lysate was directly transferred to the master mix. In automated procedures, the swab protocol of a STAR Q Swab AS instrument was used for lysis and PCR setup [8]. 2.3. PCR amplification The PCR amplifications were performed following the instructions of the Investigator 24plex QS and the Investigator 24plex GO! Handbooks [9]. Unless stated otherwise the amplifications were performed on a GeneAmp PCR System 9700 thermal cycler using a 96-well gold block. Samples were amplified in 0.2 ml thin Walled 8 Tubes or Non-skirted 96 Well PCR Plates (Thermo Fisher Scientific). The thermal cycling method for extracted DNA samples was: 3 cycles of 98  C for 30 s; 61  C for 55 s, and 72  C for 5 s, followed by

27 cycles of 96  C for 10 s; 61  C for 55 s, and 72  C for 5 s, followed by a 68  C final extension for 2 min and the 10  C soak. For cycle number testing, the 27 cycle part was reduced or increased accordingly. The thermal cycling method for direct samples was: 3 cycles of 98  C for 30 s; 61  C for 40 s, and 72  C for 5 s, followed by 22 to 24 cycles of 96  C for 10 s; 61  C for 40 s, and 72  C for 5 s, followed by a 68  C final extension for 2 min and the 10  C soak. Extracted DNA samples were amplified for 30 cycles. Unless stated otherwise, Blood on FTA and other paper were amplified for 25 PCR cycles and Buccal cells on FTA and other paper and buccal swab lysates were amplified for 27 cycles. 2.4. Sample electrophoresis and data analysis The Investigator 24plex QS and Investigator 24plex GO! Kits use a six-dye chemistry (6-FAM, BTG, BTY, BTR2, BTP, BTO). Spectral resolution was established using the Matrix Standard BT6 to allow evaluation of each fluorescence dye contained in the kit. All analyses used the DNA Size Standard 550 (BTO), and the allelic ladder provided with the kits. The amplified PCR products were separated and detected on the Applied Biosystems 3500 Genetic Analyzers (Thermo Fisher Scientific) using POP-4 polymer (Life Technologies) and a 36 cm capillary array. Injections were performed at 1,2 kV for 30s. For sample detection, a mixture of 12 ml Hi-Di Formamide (Life Technologies) and 0.5 ml DNA Size Standard 500 (BTO) per sample was set up. 12 ml of the mix were combined with 1 ml sample or 1 ml of the allelic ladder. Samples were denatured at 95  C and snap-cooled on ice for 3 min. Data analysis for initial fragment sizing and allele calling was performed using Applied Biosystems GeneMapper ID-X Software version 1.2 or 1.4 with the 24plex QS Panels, Bin sets and stutter files and a 50 RFU (relative fluorescence units) threshold unless otherwise stated. 2.5. Inhibitor stock sample preparation Humic acid (Acros) was dissolved in water at a concentration of 1.000 ng/ml. Hematin porcine (Sigma-Aldrich) was dissolved in 100 mM NaOH at a concentration of 3.500 mM. Tannic acid (SigmaAldrich) was dissolved in water at a concentration of 10.000 ng/ml. Indigo carmine (Alfa Aesar) was dissolved in water at a concentration of 30 mM. Collagen (Sigma-Aldrich) was dissolved in water at a concentration of 1.000 ng/ml. Calcium hydrogen phosphate (VWR) was dissolved in water at a concentration of 20 mM. Ethanol (96%) was used. All inhibitors were diluted to their working concentrations in water. 2.6. Concordance study DNA samples used for the concordance study were created as previously described [10]. The blood samples were extracted, quantified, and previously typed with AmpF‘STR Identifiler (Applied Biosystems), AmpF‘STR NGMSElect (Applied Biosystems), PowerPlex 16 (Promega), PowerPlex ESX 17 and ESI 17 (Promega), PowerPlex Fusion (Promega), PowerPlex Y23 (Promega). The six genomic components of Standard Reference Material (SRM) 2391c PCR-based DNA Profiling Standard were also evaluated for concordance to certified materials. A total of 656 samples were evaluated in this study for concordance testing. All genotyping was performed at NIST (National Institute of Standard Technologies) with GeneMapper ID-X v1.4 software (Applied Biosystems) using manufacturer provided allelic ladders, bins, and panels. 2.7. Stutter calculation Stutter peak analysis for Investigator 24plex QS was conducted using the 656 NIST population dataset for all loci analyzed. For

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Fig. 1. Influence of MgCl2 concentration. Fast Reaction Mix 2.0 (FRM 2.0) was supplemented with different concentrations of MgCl2 matching the specification of production, or 30% higher and lower. 500 pg of control DNA 9948 was amplified on a GeneAmp PCR System 9700 thermal cycler. Representative electropherograms of sample quadruplicates are shown. Y axis: 12,000 RFU.

Investigator 24plex GO! Kits, stutter peak heights were analyzed for 171 direct PCR samples. Here, blood and buccal cells on different FTA cards, blood on non-FTA card, buccal cells on Polyester swabs, and buccal cells on cotton swab were used. All samples were amplified using the recommended amplification protocol from the kits’ Handbooks and were analyzed on the Applied Biosystems 3500 Genetic Analyzer. Stutter peaks were determined as three bases smaller (n-3) than D22S1045, and four bases smaller (n-4) than tetranucleotide repeats (0.5 bases). Stutter positions, where heterozygous alleles differed by two repeat units and the forward stutter of the smaller allele overlapped with the backward stutter of the longer allele, were excluded from the analysis. The stutter percentage was calculated by dividing the peak height of the stutter peak by the peak height of the true allele.

3. Results and discussion 3.1. PCR-based procedures 3.1.1. Reaction conditions The final concentrations of all reaction mix components were optimized during development of the kits to ensure robust performance. In this experiment, various concentrations of MgCl2, one of the critical buffer components, were added to a standard reaction with 500 pg control DNA 9948 (Fig. 1), with blood on FTA (Fig. 2) and buccal swab samples (Fig. 3). For Investigator 24plex QS, the assay yielded robust results within a MgCl2 concentration range of 30% of the kit specification. At 30% amplification efficiency of the Quality Sensor was slightly impaired, whereas all STR markers were still unaffected. At 50% the large Quality Sensor

Fig. 2. Influence of MgCl2 concentration. Fast Reaction Mix 2.0 (FRM 2.0) was supplemented with different concentrations of MgCl2 matching the specification of production, or 15% higher and lower. Blood on FTA samples were amplified using 25 cycles on a GeneAmp PCR System 9700 thermal cycler. Representative electropherograms of sample triplicates are shown. Y axis: 5000 RFU.

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Fig. 3. Influence of MgCl2 concentration. Fast Reaction Mix 2.0 (FRM 2.0) was supplemented with different concentrations of MgCl2 matching the specification of production, or 15% higher and lower. Buccal swabs were lysed in Investigator STR Lysis Buffer and 2 ml sample was amplified using 27 cycles on a GeneAmp PCR System 9700 thermal cycler. Representative electropherograms of sample triplicates are shown. Y axis: 18,000 RFU.

fragment was not amplified and the high molecular weight markers D21S11, SE33, D2S1338, FGA and D7S820 showed partial or full drop out (data not shown). Investigator 24plex GO! yielded robust results within a MgCl2 concentration range of 15% of the kit specification. Under these conditions, amplification of the STR markers was well balanced and no dropout or non-specific amplification occurred. When MgCl2 was reduced more than 25%, again the large Quality Sensor fragment and high molecular markers were affected. Furthermore split peaks were observed in particular at D2S441 and D18S51. 3.1.2. Effect of PCR annealing temperature variations Specificity, sensitivity and robustness are affected by the annealing temperature (Tm). Since the actual Tm may vary depending on cycler conditions, the assays were validated in a range surrounding the temperature given in the kit manual (first 3 cycles at 64  C, following 27 cycles at 61  C). For Investigator 24plex QS, annealing temperatures between 4  C and +4  C around the annealing temperature of 64  C/61  C were applied to the amplification of 500 pg control DNA 9948. For Investigator 24plex GO! annealing temperatures between 4  C and +3  C were applied to the amplification of blood on FTA, and buccal swab samples. The annealing temperature for the first 3 cycles and for the following 27 cycles was varied to the same extent. PCR was performed on an Eppendorf1 Mastercycler1 ep instrument in triplicates. For Investigator 24plex QS, reactions using annealing temperatures between 4  C and +4  C resulted in full profiles. The highest overall peak heights were observed with annealing temperatures of 64  C/61  C. In the temperature range of 3  C to +1.5  C, no marker showed a peak height difference of more than 50% to the average profile height. At +3  C DYS391, D10S1248, D12S391, and D22S1045 showed the most significant peak height reduction; all were reduced to about 30% of the height at the standard protocol annealing temperature. For Investigator 24plex GO!, reactions using annealing temperatures between 4  C and

+3  C resulted in full profiles. However, the average peak height of markers was highest for the conditions closest to the actual annealing temperature of 64  C/61  C. As expected, the markers most susceptible to increased annealing temperatures were the same for both assays. No dropouts were observed in the tested range for both kits applying a threshold of 100 RFU. No non-specific PCR products were observed. 3.1.3. Effect of different cycle numbers PCR cycle numbers can be altered to adapt the reaction conditions to varying DNA template concentrations. Cycle numbers can be either increased to enhance amplification signals when working with low-copy-number DNA, or decreased to speed up the protocol when the DNA sample is abundant. In particular for direct amplification the amount of sample material present in a reaction varies between different reference sample types, donors, collection procedures and storage conditions. Therefore, for optimal results it is important to evaluate a representative batch of samples and to adapt reaction conditions if necessary. For the Investigator 24plex QS Kit, the cycle numbers were increased to 32 or 34 for reactions containing 32, 16 or 8 pg of control DNA 9948, and the numbers of called alleles, as well as peak heights, were compared to a standard 30-cycle protocol (Fig. 4). Here, the cycle numbers of the second cycling block were increased from 27 cycles to 29 or 31, while the first 3 cycles of the standard protocol were not changed. As expected, signal intensities of amplified products increased with higher cycle numbers. However, it should be noted that an increase in overall cycle number to more than 30 will not necessarily result in more information obtained from the low-template-DNA sample. Furthermore, because of stochastic effects, increased peak imbalances or dropouts may in general be observed for low-copy-number samples (with 100 pg or less of template DNA), regardless of any increase in cycle numbers. In this study, using a threshold of 50 RFU for allele calling, the number of allelic dropouts due to stochastic

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Fig. 4. Effect of different cycle numbers on mean peak height and number of called alleles. Different amounts of control DNA 9948 were used as template, as indicated in the figure. Samples were run in triplicates and numbers of detected PCR products (indicated above the bars; 120 PCR products expected) and their peak heights were calculated. 50 RFU was used as a threshold for detection.

Fig. 5. Effect of decreased cycle numbers on mean peak heights. 2.5 ng or 10 ng of control DNA 9948 were subjected to amplification using a total of 24, 26 or 28 PCR cycles. Samples were run in triplicates and average peak heights calculated.

effects was not significantly reduced when more PCR cycles were applied. In particular, for reference samples such as buccal swabs, where DNA can be extracted in abundance and increased template amounts result in very high heterozygote balance, reduced cycle numbers may be used to streamline the laboratory workflow. Here, cycle numbers were decreased to 24, 26 or 28, for reactions containing 2.5 ng or 10 ng of template DNA (Fig. 5). The cycle numbers of the second cycling block were decreased from 27 to 21, 23 or 25, while the first 3 cycles of the standard protocol were not changed. As expected, all reactions resulted in robust amplification and full profiles were obtained using a threshold of 50 RFU for allele calling. However, amplifications with a total of 28 total cycles, using 10 ng template DNA, gave rise to pull up peaks when applying samples to analysis without prior dilution of the PCR product. Please note, when reducing the cycle number, the peak heights of the Quality Sensor QS1 and QS2 decrease and may drop below the threshold. The template amount of the Quality Sensor in the Investigator 24plex QS Kit is optimized for 30 cycles, thus reducing the cycle number will reduce the QS signals. Hence, in the case of reduced cycle numbers the QS signals will not give any information about an inhibited PCR or degraded template DNA. For Investigator 24plex GO!, blood or buccal cells on FTA and buccal swab lysates were amplified using the cycle number recommended as starting point for evaluation, +/ two PCR cycles (Fig. 6). Samples from three donors were analyzed in five replicates

Fig. 6. Effect of different cycle numbers on mean peak height of reference samples subjected to direct amplification. Samples were amplified in five replicates per donor on a GeneAmp PCR System 9700 thermal cycler. (A) Blood on FTA paper. (B) Buccal cells on FTA paper. (C) Buccal swab lysates.

each. As expected, average signal heights in most cases increase with each cycle added. Buccal cells on FTA show considerable sample to sample differences caused by inhomogeneous deposition of cells during sample transfer. This is reflected by the fact that the expected increase in signal with higher cycle numbers is not

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Thermal Cycler (Applied Biosystems Inc., Foster City, CA, USA), Eppendorf Mastercycler ep (Eppendorf AG, Hamburg, Germany), MJ Research DNA Engine1 PTC-200 Peltier Thermal Cycler (BioRad Laboratories GmbH, Munich, Germany). Comparable mean peak heights were obtained for all of the tested PCR cyclers (Figs. 7 and 9). No significant differences in intra- and inter-locus balance or non-specific amplification were observed on any of the thermal cyclers (Fig. 8). Only samples with at least 500 pg template were analyzed for inter-locus balance, because of stochastic effects present with the lower DNA input amounts used to compare sensitivity. Fig. 7. Investigator 24plex QS performance on different PCR thermal cyclers. Different amounts of control DNA 9948 were used as indicated in the figure. Each sample was run in duplicate. A standard 30-cycle protocol was used. Average heterozygous peak heights across all markers are shown.

observed in all cases (see Fig. 6, donor 1 as example). The Quality Sensor template and primer concentrations were optimized during development to provide stable peak heights across the PCR cycle number range typically applied. 3.1.4. Effect of different cycler types Usually, not all forensic laboratories are using the same thermocyclers. To demonstrate the kits’ robustness independent of the instrument, several PCR cyclers were tested with the Investigator 24plex QS Kit and the Investigator 24plex GO! Kit. 2 ng to 32 pg of control DNA 9948 were used as PCR template for Investigator 24plex QS. Investigator 24plex GO! used blood on FTA and buccal swab lysates from five different donors which were run in four replicates each. The reaction took place under standard conditions using 30 cycles (Investigator 24plex QS), 25 PCR cycles for blood on FTA and 27 cycles for swab lysates (Investigator 24plex GO!)), and was performed with the following thermal cyclers: GeneAmp PCR System 9700 with Aluminum 96-Well Block (Applied Biosystems Inc., Foster City, CA, USA), GeneAmp PCR System 9700 with Silver or Gold-plated Silver 96-Well Block (Applied Biosystems Inc., Foster City, CA, USA), Veriti1 96-Well

3.2. Sensitivity To test sensitivity and robustness towards DNA template input of the Investigator 24plex QS Kit, Control DNA 9948 was serially diluted from 1 ng to 8 pg per reaction. A 50 RFU threshold was used for allele designation. 100% of the alleles were consistently obtained at 125 pg, using the standard conditions specified in the kit handbook. Occasional allele dropouts were found due to stochastic effects when less than 63 pg DNA were used as template. For 32 pg 98%, for 16 pg 81% and for 8 pg 50% of all expected alleles were called. As expected, the number of dropouts increased with decreasing DNA concentration without any specific pattern for a certain locus. Heterozygous peak height ratios decreased towards lower template amounts (Fig. 10). No saturation was observed with 1 ng DNA. The sensitivity of Investigator 24plex GO! Kit was tested using direct amplification samples. Dilution series of blood were spotted on FTA paper and amplified using 25 PCR cycles. Swab lysates were diluted and amplified using 27 PCR cycles. For both sample types, 3-fold dilutions down to 1:81 were used. The obtained signal heights correlated well with the dilution factor of the sample material (Fig. 11). Down to 1:81 fold dilutions all samples provided full profiles for buccal cell lysates. Using blood on FTA as template, all samples provided full profiles for 1:3 fold dilutions. For 1:9 fold and higher dilutions, increasing numbers of alleles dropped below the 50 RFU thresholds. Note that in these cases profiles can be recovered by increasing PCR cycle numbers (data not shown).

Fig. 8. Intracolor peak height ratios for each dye channel. 500, 1000 and 2000 pg samples were analyzed resulting in 6 samples per cycler investigated.

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Fig. 9. Investigator 24plex GO! performance on different PCR thermal cyclers. Average heterozygous peak heights of four replicates across five different donors are shown. Error bars show the standard deviation between all replicates and donors.

3.3. Sensitivity in the context of the Quality Sensor The Investigator 24plex QS and Investigator 24plex GO! Kit contain an internal PCR control which is amplified as a small amplicon Quality Sensor peak (QS1) at 72 bp and large amplicon Quality Sensor peak (QS2) at 435 bp depending on the performance of the PCR or the quality of DNA. The primers and an artificial DNA template for the Quality Sensor are enclosed in the Primer Mix, and are amplified simultaneously with the polymorphic STR markers. To analyze the effect of the Quality Sensor on the sensitivity, control DNA 9948 was serially diluted from 1 ng to 8 pg per reaction, and analyzed with and without the amplification of the Quality Sensor. The reactions were performed in quadruplicates and mean values of peak heights (Fig. 12) and the called alleles of all markers were calculated. Full profiles (160/160 alleles) were consistently obtained at 125 pg for both conditions, with or without the Quality Sensor. Occasional allele dropouts were found

due to stochastic effects when 64 pg DNA was used as template. As expected, the number of dropouts increases with decreasing DNA concentration. Again, no statistical relevant difference between both approaches with or without the Quality Sensor was observed for template amounts, when 64 pg DNA was used as template. 3.4. Performance with simulated inhibition To test the robustness of the Investigator 24plex QS Kit, the assay was run in the presence of the inhibitors, chosen to mimic challenging forensic sample types. Inhibitors tested were humic acid (50–250 ng/ml), a principal component of humic substances that may be co-extracted from forensic samples collected from soil, hematin (250–1250 mM), formed by the oxidation of heme, the main component of blood, tannic acid (500–4000 ng/ml), typically present in leather, indigo carmine (4–12 mM), the dye of blue

Fig. 10. Sensitivity study for Investigator 24plex QS. Serial dilutions of control DNA 9948 were analyzed. The amounts of DNA indicated were used as template for amplification. Each template amount was analyzed in 4 replicates. The percentage of alleles called (grey) and heterozygous peak height ratio (black) are shown.

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Fig. 11. Sensitivity study for Investigator 24plex GO!. Serial dilutions of blood samples on FTA cards or buccal swab lysates were analyzed in 5 replicates each.

denim, collagen (100–300 ng/ml), the main protein compound of many tissues, calcium (1–5 mM), released during lysis of bones, and ethanol (0.1–4%), a potential carryover of the DNA extraction method. As template, 500 pg of control DNA 9948 were used in duplicates and PCR was performed under standard conditions. As a threshold for allele calling, 50 RFU was used. Results from the study can be found in Fig. 13. With humic acid, full profiles were generated in presence of 200 ng/ml, with 64% of alleles still present at 250 ng/ml. For hematin full profiles were generated in presence of 750 mM, with 75% of alleles still present at 1000 mM. The patterns of markers that are most affected by hematin and humic acid were very similar, which may suggest a common mechanism. With tannic acid, full profiles were generated for all concentrations tested (up to 4000 ng/ml final concentration in the PCR). For indigo carmine consistently full profiles were obtain for all concentrations tested with D18S51 showing split peaks at the highest concentration of 12 mM. For collagen, full profiles were generated with 150 ng/ml. At 200 ng/ml, whilst full profiles were obtained, split peaks were observed, most prominently at D18S51, D2S441, D10S1248, D22S1045 and CSF1PO. With calcium, full profiles were generated in the presence of 3 mM. Only one allele dropped

out at 4 mM, but split peaks were again found with the same markers most affected as for collagen. The fact that both, collagen and calcium, show pronounced split peak formation indicates these inhibitors primarily act on the Taq polymerase. This is in agreement with findings from other studies [11]. When testing ethanol, full profiles were generated in presence of 2% with split peaks at D2S441, D10S1248, and D22S1045. Addition of 4% ethanol led to almost full inhibition. As expected, a gradual reduction in peak heights can typically be observed at concentrations lower than those resulting in allele drop out. For all inhibitors studied here, the large Quality Sensor fragment (QS2) was most susceptible to inhibition. For Investigator 24plex GO! robustness towards inhibitors was tested by applying increasing sample amounts. Here, potential inhibitors are contained in the sample itself, e.g. as components of FTA paper, or hematin. One, two or three punches of 1.2 mm diameter from blood or buccal cells on FTA paper were used as sample. Buccal cells on FTA paper were amplified with the additive Investigator STR GO! Punch Buffer, as recommended in the Handbook. This buffer helps to overcome inhibition caused by FTA paper components. Buccal swabs were prepared with the Investigator STR GO! Lysis Buffer, and 1, 2 or 4 ml of crude lysates were used. For all sample types triplicates were run. For blood on FTA, consistent full profiles were obtained for all samples. When 3 punches were amplified, lower peak heights were observed for high molecular weight markers in one sample. Here, QS2 in addition indicated low-level inhibition. For buccal cells on FTA, very similar results were obtained. In particular, introduction of three punches of blood on FTA frequently results in reduced amplification of the longer STR markers and of the Quality Sensor QS2, as well as shoulder formation of marker D18S51. These effects indicate inhibition of the PCR by the increased amount of FTA paper. With increasing sample input of buccal cells on FTA paper the overall signal intensity escalates. Introduction of three punches of FTA might lead to marginal inhibition effects. For buccal swabs, all tested input volumes gave rise to full profiles, but profile balance may be negatively affected when using 4 ml lysate as sample input.

Fig. 12. Effect of the Quality Sensor on sensitivity. Serial dilutions of Control DNA 9948 were analyzed in PCR reactions with and without the Quality Sensor template. The experiments were performed in quadruplicates. On the X axis, the indicated amounts of DNA were used as template for amplification. Average peak heights are shown.

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Fig. 13. Overview of Investigator 24plex QS inhibitor resistance. The assay was tested for its robustness towards inhibitors (humic acid, hematin, tannic acid, indigo carmine, calcium, collagen, and ethanol). 500 pg of control DNA 9948 was used as template and PCR was performed under standard conditions. 50 RFU was used as a threshold for allele calling. Green: Consistently full profile. Yellow: 75% of expected PCR products detected. Orange: 50% of expected PCR products detected. Red: Less than 50% of expected PCR products detected. Light Green: Consistently full profile with split peaks. Shaded: Average peak heights more than 50% reduced. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.5. Stability and efficiency

3.6. Case-type samples

To prove stable results after multiple rounds of freezing and thawing, 500 pg DNA 9948 were used as template DNA in triplicates with fresh kit components (no freeze/thaw) and with kit components stressed by 20 rounds of freezing and thawing (20x freeze/thaw). The overall kit performance was not compromised under the chosen conditions. 100% of the alleles were called for both setups with comparable peak heights obtained before and after 20 rounds of freezing and thawing (data not shown).

A total of 87 mocked case-type samples were analyzed. The samples were chosen to represent the typical variation in DNA content, integrity and potential presence of inhibitors. Surface swabs were taking from a computer keyboard and mouse, and from the inner surface of gloves (total 35 samples). Cigarette butts (24 samples) and chewing gums (12 samples) were collected from different donors and a fraction was exposed to environmental conditions to force DNA degradation. In order to mimic inhibitory

Fig. 14. Example of an exposed cigarette butt sample profile. The Quality Sensor fragments are labeled. Y Axis: 6000 RFU.

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samples, blood was applied to fabric contaminated with used engine oil and to swabs soaked in mud (total of 16 samples). All profiles obtained were concordant with the expected profile of the major donor. All cigarette butt, chewing gum and blood stain samples gave rise to full profiles. The exposed cigarette butts showed ski slope like profiles indicative of DNA degradation. None of the blood stains resulted in any obvious inhibition, indicating the inhibitors were removed during extraction of DNA. 31 of the surface swab samples gave rise to full profiles, 4 to low level partial profiles. A majority showed DNA degradation to various degrees. For all affected samples, degradation as cause of the reduced peak heights of long amplicon markers was supported by a balanced amplification of the two Quality Sensor fragments (Fig. 14). 3.7. Precision Precise sizing of amplified fragments is crucial for determination of correct genotypes. To measure the degree of variation to be expected, a full plate of 96 allelic ladders was run on a 3500 Genetic Analyzer. Average length and standard deviations were determined for each ladder fragment. As expected, variation increases with larger fragment sizes (Fig. 15). The maximum standard deviation was 0.08 bases observed for SE33 allele 33.2. The degree of variation is similar to that reported in other studies [12]. 3.8. Reproducibility To test for reproducibility of genotyping results across different sites, operators, and instruments, Control DNA 9948 was analyzed at three sites by different operators in 8 replicates. 3500 (all three sites), or 3130 Genetic Analyzers (one site) were used. All genotypes obtained were identical and concordant with known values. 3.9. Mixture study To assess the ability to resolve minor contributor alleles with Investigator 24plex QS Kit a study was performed with 9 mixture samples at differing ratios of male:female DNA 9948 and XX107 (1:1, 3:1, 7:1, 10:1, 15:1 and vice versa). The total amount of mixed DNA used in this study was 500 pg; a 15:1 mixture thus contains 31 pg of the minor component DNA and 469 pg of the major component. Samples were run in replicates of 4. The limit of detection of the minor component was determined by analyzing non-overlapping alleles of both DNAs. Fig. 16 summarises all data generated. 100% of expected alleles were identified for minor components of 3:1, 7:1 and 10:1 mixtures. For 15:1 97% of the minor component alleles were identified. This result is in line with

Fig. 15. 96 allelic ladders were run on a 3500 Genetic Analyzer and data analyzed using GeneMapper IDX v1.2.

Fig. 16. Mixture analysis. Two source mixtures of ratios 15:1 to 1:15 were prepared and amplified using 500 pg template DNA. Amplification was performed according to the recommended protocol. Samples were analyzed using the Applied Biosystems 3500 Genetic Analyzers applying a 50 RFU allele call threshold.

the sensitivity study, which showed drop outs to occur when 32 pg DNA were analysed. A mixture studies for Investigator 24plex GO! was not performed due to the nature of reference samples. 3.10. Concordance NIST genotyped DNA samples from 656 unrelated individuals (NIST U.S. population set (650 samples) and SRM 2391c (6 samples) with the Investigator 24plex QS Kit to demonstrate concordance. Since the Investigator 24plex GO! Kit contains the same primer sequences as the Investigator 24plex QS Kit, the results of the NIST concordance study are valid for both kits. The study’s results were aligned with genotypes generated previously with other STR systems. Out of 29,520 alleles compared, one discordant call was observed resulting in a 99.997% concordance between the Investigator 24plex QS and the NIST final data set. No null alleles were observed. The only discordant sample generated an 8, 9.3 genotype at D7S820 with Investigator 24plex QS as well as AmpF‘STR1Identifiler, AmpF‘STR1 Profiler Plus and PowerPlex Fusion. All other assays (Powerplex 16, AmpF‘STR MiniFiler, Investigator IDplex Plus) gave an 8, 11. The cause of this discrepancy was confirmed by sequencing. A 5 bp deletion 114 bp downstream from the repeat in the Investigator 24plex QS amplicon is outside the amplified region of other STR assays, e.g. Investigator IDplex Plus [13]. 3.11. Stutter Slippage events are known to occur when repetitive STR sequences are amplified by PCR [14], and these events result in stutter. Stutters having one repeat unit less than the corresponding allele peak are most frequent and therefore reported here. Called stutter peaks might lead to data misinterpretation and must be taken into consideration for most samples. In this study, the stutter peak analysis for Investigator 24plex QS was conducted using the NIST population dataset for all loci analyzed. For Investigator 24plex GO! Kits, stutter peak heights were analyzed for 171 direct PCR samples (57 blood on FTA card (Whatman FTA Micro Card), 38 blood on FTA card (Whatman EasiCollect), 19 blood on non-FTA card (903 Protein Saver Snap-Apart Card, Whatman), 19 buccal cells on FTA card (Whatman EasiCollect card), 19 buccal cells on Polyester swab (Puritan Sterile Cotton tipped Applicators), and 19 buccal cells on cotton swab (Sarstedt Forensic Swab). Each sample was analyzed in 5 replicates. Smaller alleles display lower stutter levels than longer alleles of the same marker. Tables 1 and 2 summarizes the percentage of back stutters for each locus per multiplex kit. Stutter peak heights are characteristic for each

M. Kraemer et al. / Forensic Science International: Genetics 29 (2017) 9–20 Table 1 Stutter ratios observed for the Investigator 24plex QS Kit. System

Observations Stutter Mean (%) Stutter Min (%) Stutter Max (%)

CSF1PO D10S1248 D12S391 D13S317 D16S539 D18S51 D19S433 D1S1656 D21S11 D22S1045 D2S1338 D2S441 D3S1358 D5S818 D7S820 D8S1179 DYS391 FGA SE33 TH01 TPOX vWA

825 889 1019 869 849 1012 934 1026 1003 885 1066 890 885 797 850 895 608 1017 1132 787 827 909

6.2 8.2 8.9 5.5 6.6 8.5 6.8 9.1 7.4 8.6 8.2 5.4 8.6 5.8 4.8 7.0 6.2 7.6 9.7 2.9 3.3 7.0

2.0 3.1 3.6 1.5 2.6 3.0 3.0 2.9 2.5 1.6 3.4 0.8 3.2 1.3 1.5 2.7 3.0 1.8 2.6 1.1 1.1 0.8

11.9 14.3 17.6 11.4 12.2 17.2 14.2 16.5 12.8 19.9 14.2 11.1 15.1 11.5 9.6 12.4 9.6 14.8 19.9 6.5 7.9 12.8

System

Observations Stutter Mean (%) Stutter Min (%) Stutter Max (%)

CSF1PO D10S1248 D12S391 D13S317 D16S539 D18S51 D19S433 D1S1656 D21S11 D22S1045 D2S1338 D2S441 D3S1358 D5S818 D7S820 D8S1179 DYS391 FGA SE33 TH01 TPOX vWA

967 997 1445 1200 1052 1289 1107 1429 1341 1181 1301 1412 1110 982 1119 1093 402 1246 1565 1172 1115 1115

2.3 5.6 3.7 1.6 2.2 3.3 2.3 4.7 3.9 1.2 2.8 1.5 3.6 2.5 0.8 2.6 4.4 2.9 3.2 0.7 1.6 0.5

general shows the lowest stutter ratio of all STR markers of the Investigator 24plex QS and Investigator 24plex GO! Kit. Forward stutters are usually of very low height, and are not shown for this reason. Please note that marker D22S1045 shows a significantly elevated forward stutter. This is intrinsic and due to the fact that the marker consists of trinucleotide instead of tetranucleotide repeats. This may also lead to unexpectedly high back stutter peaks if alleles differ by two repeat units and the forward stutter of the smaller allele overlaps with the back stutter of the longer allele. 3.12. Species specificity

Table 2 Stutter ratios observed for the Investigator 24plex GO! Kit.

5.9 8.4 9.3 4.9 5.8 7.5 6.4 8.7 7.3 6.4 7.6 4.8 8.9 5.7 4.1 6.7 6.1 7.0 9.0 2.0 3.2 6.5

19

11.0 13.5 17.4 12.3 12.6 14.9 10.7 15.2 12.3 13.4 14.6 11.8 17.0 9.6 9.7 12.7 8.3 13.1 15.8 4.9 7.8 11.9

Non-human DNA can be present in forensic casework samples. To verify Investigator 24plex QS Kit species specificity for human DNA, DNA from other species was tested. Besides common pets and farm animals with 2.5 ng of sample input, some primates were also tested with 500 pg sample input. Table 3 summarizes the testingsetup and results for each species tested in duplicates. As expected for primates, amplification of some products is possible. Chimpanzees, bonobos, orangutans and gorillas give rise to several peaks within marker ranges in all channels, some of which match the size of human STR products. The profiles obtained from primates can be distinguished from human profiles by the presence of many off-ladder peaks and the overall imbalance. Similar findings have been reported in previous studies [16]. For Investigator 24plex QS, Macaque DNA produced an Amelogenin Xpeak, one allele call for D1S1656 allele 12 and further off-ladder peaks in the FAM, BTG and BTR panel. Most of the further tested animal DNAs did not show any cross reactivity with the kits. Using 2.5 ng sheep DNA as template, three or less off-ladder peaks (<50 RFU) were produced in the BTG channel. Dogs showed one allele call (<60 RFU) for D2S441, allele 14. The Investigator 24plex GO! Kit contains the same primer sequences as the Investigator 24plex QS Kit, the results of the species specificity study are thus valid for both kits. 4. Conclusion

marker and the number of repeat motifs of an allele [15]. The default locus-specific stutter filter values included in the stutter filters are based on all stutters observed in this study. TH01 in

The Investigator 24plex QS Kit and Investigator 24plex GO! Kit were developed for STR genotyping and human identification. In this study the performance of the kits was tested for the following parameters: Component concentrations, cycling protocol, sample input amounts and sensitivity, robustness and stability, reproducibility, precision, cross reactivity, mixtures, and stutter. Experiments were designed to reflect the application the kits have been developed for. A concordance study was conducted using an U.S. population set. The results demonstrate the reliability of results obtained with the Investigator 24plex QS Kit and Investigator 24plex GO! Kit in accordance with the ENFSI and SWGDAM validation guidelines [3,4]. It was shown that the Quality Sensor included in the assays provides information on the quality and integrity of samples analyzed. The study gives context for

Table 3 Unspecific amplification products found with other species. Species

Amount tested

Investigator 24plex QS Kit Peak >50 RFU

Chimpanzee, bononbo, orangtutans, gorillas Macaque Dog Cat Horse Pig Cow Sheep Goat

500 pg 500 pg 2,5 ng 2,5 ng 2,5 ng 2,5 ng 2,5 ng 2,5 ng 2,5 ng

Various, most products are from STR markers AM X, D3S1358 OL, D1S1656 allele 12, D2S441 allele 19 D2S441, allele 14 no no no no no no

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laboratories for internal validations they should perform with the procedures and instrumentation established. Conflict of interest None. Acknowledgments The authors would like to gratefully acknowledge the members of the Human Identification R&D, Marketing, and Operations groups (QIAGEN GmbH) for technical assistance and valuable comments on this work. References [1] D. Hares, Selection and implementation of expanded CODIS core loci in the United States, Forensic Sci. Int. Genet. 17 (2015) 33–34. [2] D. Hares, Expanding the CODIS core loci in the United States, Forensic Sci. Int. Genet. 6 (2012) e52–e54. [3] ENFSI Standing Committee for Quality and Competence (QCC). (2006) Validation and Implementation of (New) Methods. Ref. Code: QCC-VAL-001, Issue No. 001, November 2006. http://www.enfsi.eu/get_doc.php?uid=144. [4] Scientific Working Group on DNA Analysis Methods (SWGDAM), Validation Guidelines for DNA Analysis Methods, December 2012. http://media.wix.com/ ugd/4344b0_cbc27d16dcb64fd88cb36ab2a2a25e4c.pdf. [5] C. Phillips, L. Fernandez-Formoso, M. Garcia-Magariños, L. Porras, T. Tvedebrink, J. Amigo, M. Fondevila, A. Gomez-Tato, J. Alvarez-Dios, A. Freire-Aradas, A. Gomez-Carballa, A. Mosquera-Miguel, A. Carracedo, M.V. Lareu, Analysis of global variability in 15 established and 5 new European

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