Technical Note. An evaluation of the precision of QIAGEN Quantiplex HYres kit as a screening tool on low level touch DNA evidence

Technical Note European Journal of Forensic Sciences www.ejfs.co.uk DOI: 10.5455/ejfs.19657 An evaluation of the precision of QIAGEN Quantiplex HYres kit as a screening tool on low level touch DNA evidence Stephen Chi-Yuen Ip, Kam-Ming Lai Forensic Science Division, Government Laboratory, Homantin Government Offices, Kowloon, Hong Kong, China Address for correspondence: Stephen Chi-Yuen Ip, Forensic Science Division, Government Laboratory, Homantin Government Offices, Kowloon, Hong Kong, China. E-mail: kmlai@ govtlab.gov.hk Received: July 15, 2015 Accepted: October 07, 2015 Published: November 02, 2015 ABSTRACT Objective: The primary aim of DNA quantification in forensic DNA analysis is to determine and optimize the amount of DNA for the downstream profiling analysis. In addition, it may be used as a parameter for the cut-off of the subsequent profiling, especially for touch DNA samples collected from property crime scenes, which often contain low amount of DNA and yield no profiles. The choice of quantification kit should have both high accuracy and precision. This work aims to examine the latter of five selected commonly used real-time quantitative polymerase chain reaction (qpcr) kits, namely Applied Biosystems Quantifiler kit, Quantifiler Y kit, Quantifiler Duo kit, Promega Plexor HY system, and QIAGEN Quantiplex HYres kit, and to seek for a qpcr system fitted for the purpose of initial screening of touched DNA evidence for profiling. Materials and Methods: In this work, we first tested the precision of the five selected qpcr kits using their respective DNA standard and human blood samples as substrates. Subsequently, we compared the capability of Quantiplex in predicting the presence of DNA with that of Quantifiler when applying a cut-off threshold of 0.0049 ng/μl and 0.023 ng/μl, i.e. their respective lowest detection limit with reference to DNA typing analysis. Results: Our results demonstrated that Quantiplex exhibits a higher precision and consistency than the others, especially at low DNA concentration, i.e. 0.016-0.023 ng/μl. We also showed that, using a total of 332 simulated touch DNA samples, Quantiplex is a fairly reliable predictor of the AmpFlSTR Identifiler profiling results. Conclusion: Quantiplex can be employed as a cost-effective screening of samples of volume crime cases. KEY WORDS: Forensic sciences, forensic genetics, DNA quantification, DNA typing, Plexor, Quantifiler, Quantiplex INTRODUCTION Forensic DNA analysis of crime scene samples usually involves five steps, including DNA extraction, quantification of extracted DNA, polymerase chain reaction (PCR) amplification of short tandem repeats (STR), capillary electrophoresis of amplified PCR products, and data interpretation; among which DNA quantification is essential in the overall DNA profiling process as it allows both an assessment of DNA extraction efficiency and a determination of how much DNA template to be added into the downstream analysis [1]. Typical optimum range of total input DNA lies between 0.5 and 2 ng for most of the commercially available STR amplification kits. Too much DNA template in a PCR reaction results in overblown signals in the electropherogram leading to shift in molecular size of detected signal as well as artifacts, whereas too little DNA leads to allelic drop-out and loss of information owing to stochastic effect, which happens due to random sampling of template DNA during the first few PCR cycles. These unfavorable conditions could generate unreliable results which will significantly interfere the data interpretation and impede the overall efficacy of DNA analysis. Hence, quantification of extracted DNA is indispensable to ensure PCR amplifications performed under an efficient and optimized condition. Until now, a number of DNA quantification methods, including ultraviolet absorbance, slot blot, PicoGreen, and real-time quantitative PCR (qpcr), have been employed in the forensic field to estimate the concentration of the input DNA [1-4]. qpcr is the preferred technique among these methods for forensic samples collected from scenes, as it is highly sensitive, automatable, and most importantly in human-specific. It also offers a broad dynamic range of quantification. qpcr not only identifies the presence of PCR inhibitor(s) in the samples which will affect STR amplification, but also provides essential information on whether sufficient amount of human DNA can be recovered for subsequent downstream analysis. Although qpcr analysis allows a higher sensitivity over the other quantification methods, it is nevertheless strongly affected by Eur J Forensic Sci Apr-Jun 2016 Vol 3 Issue 2 61

the stochastic effect that is very common in most PCR-based methods. of low level DNA samples often yield false negative quantification results [5,6], i.e. samples with quantification values below the lowest standard but produce informative STR profiles. Hence, this prompts the needs to look for a DNA quantification kit with both high precision and reliability, allowing forensic scientists to have a more accurate prediction on the STR amplification results and to formulate a cut-off threshold from the quantification results to decide which samples will be proceeded for further testing. In this study, we have explored the precision of several commercially available qpcr kits commonly used in forensics, including Applied Biosystems Quantifiler (refer to as Quantifiler ) [5,7-8], Quantifiler Y (refer to as Quantifiler Y ) [7], Quantifiler Duo Human DNA kits (refer to as Quantifiler Duo ) [9,10], Promega Plexor HY System (refer to as Plexor ) [11], and QIAGEN Investigator Quantiplex HYres Kit (refer to as Quantiplex ) [12]. There are several differences between these qpcr quantification kits [Table 1]. First, the chemistry for detection is different. The Quantifiler series and Quantiplex utilize Taqman assay and Scorpion primerbased assay, respectively, whereas Plexor employs a primer quenching assay. This means that the fluorescence signal from the TaqMan probes or Scorpion primers increases as PCR proceeds, whereas signal from the Plexor primers drops as a result of quenching. Because Plexor measures the accumulation of product by a decrease in fluorescence signal, an extra tailor-made program by the manufacturer is required for the analysis of preliminary data collected, and hence lengthening the total processing time. Second, the size of their target regions and copy numbers in the human genome are different. The Quantifiler series target single copy genes in the genome, whereas both Plexor and Quantiplex target multi-copy genes. Quantifiler and Quantifiler Y amplify a 62 bp and a 64 bp region on chromosome 5 and the Y chromosome, respectively [13]. Since the targets for these two kits are smaller than those of the STR amplification kits in the subsequent step, i.e. usually between 100 and 450 bp, degraded samples with positive quantification result does not necessarily guarantee the generation of DNA profile. To tackle this problem, the target size for Quantifiler Duo, Plexor, and Quantiplex are all increased to the range similar to the STR Table 1: Details of the five real-time quantitative PCR kits tested in this study kit Detection Target copy no. Target size (Chr.) Reaction time Quantifier Taqman probes Single 62 bp (5) ~110 min Quantifiler Y Taqman probes Single 64 bp (Y) ~110 min Quantifier Duo Taqman pr obes Single 140 bp (14) 130 bp (Y) ~90 min Plexor Primer quenching Multi 99 bp (17) 133 bp (Y) ~60 min Quantiplex Scorpion primers Multi 146 bp (several) ~60 min 129 bp (Y) Chr. indicates the chromosome where the target is located. PCR: Polymerase chain reaction kits. Quantifiler Duo amplifies a 140 bp and a 130 bp target on chromosome 14 and the Y chromosome, respectively [14], whereas Plexor amplifies a 99 bp and a 133 bp multi-copy target on chromosome 17 and the Y chromosome, respectively [15]. Quantiplex, on the other hand, targets a proprietary region that is a 146 bp that are present in several autosomal regions of the human genome, and a 129 bp multi-copy fragment on the Y chromosome. Hence, with these adjustments, these kits are claimed to offer a greater sensitivity and a higher reliability within different individuals and populations [16]. In addition, Quantifiler Duo [9,17], Plexor [11], and Quantiplex [12,17] that are capable of quantifying autosomal and male DNA simultaneously, all offer further information on the relative quantities of human male to female DNA in a sample that will facilitate the selection and implementation of appropriate downstream methods for genotyping analysis. The first objective of this study was to compare the precision and consistency of the five selected qpcr kits, viz., Quantifiler, Quantifiler Y, Quantifiler Duo, Plexor, and Quantiplex. In addition, it was aimed to seek for a qpcr system fitted for the purpose of initial screening of touched DNA evidence for profiling. MATERIALS AND METHODS Preparation of DNA Standard Samples Series of DNA standards of each quantification kits were prepared freshly prior to the experiment according to the manufacturer s recommendations to avoid any deterioration of the quality of standards in the course of the experiments. The precision of a real-time qpcr method was examined by performing three runs on different days using the standard concentrations (refer to as Standard ; in ng/μl) in the range of 2.00-5.56, 0.31-0.62, 0.016-0.023 and 0.0032-0.0049 ng/ μl for autosomal DNA [Table 2], and 1.66-5.56, 0.10-0.62, 0.006-0.023, and 0.0016-0.0032 ng/μl for male DNA [Table 3]. Each run contained 10 replicates of the selected standard concentrations (n = 30). Preparation of Whole Blood DNA Sample Whole blood samples were collected from two female and two male volunteers, respectively, with consent. The extraction of DNA from the whole blood samples were performed using the QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany) [18] following the manufacturer s recommendations. DNA was eluted in approximately 200 μl of Buffer AE. The extracted whole blood DNA samples were diluted to approximately 0.5 and 0.025 ng/μl with Trisethylenediaminetetraacetic acid buffer as estimated with Quantifiler. These samples, which were defined as High and Low range, respectively, were stored at 20 C before analysis. The precision of a real-time qpcr method was examined by performing three runs on different days on the whole blood DNA. Each run contained 10 replicates of samples (n = 30). 62 Eur J Forensic Sci Apr-Jun 2016 Vol 3 Issue 2

Table 2: The relationship between the cycle threshold (C T ) and its standard deviation with the concentration of the DNA template when autosomal DNA was targeted (n=30) Standard concentration (ng/μl) kit 2.00-5.56 0.31-0.62 0.016-0.023 0.0032-0.0049 Quantifiler 5.56 25.9-27.4 26.3 (±1.2) 0.62 28.9-29.5 29.2 (±0.6) 0.023 32.6-35.3 33.6 (±1.9) NA NA NA Quantifier Duo 5.56 26.1-27.2 26.7 (±1.3) 0.62 29.3-30.2 29.8 (±1.0) 0.023 32.9-37.9 34.3 (±2.8) NA NA NA Plexor 2.00 21.3-22.7 22.0 (±2.2) 0.40 22.7-25.4 24.4 (±2.1) 0.016 26.8-31.0 29.2 (±3.0) 0.0032 29.8-33.3 31.4 (±3.2) Quantiplex 5.00 23.5-24.1 23.8 (±0.8) 0.31 27.2-28.0 27.6 (±0.8) 0.019 30.6-31.7 31.2 (±0.9) 0.0049 28.0-35.5* 33.2 (±3.4) NA indicates DNA quantification is not applicable for Quantifiler and Quantifiler Duo in the range of 0.0032-0.0049 ng/μl. *Indicates the detection of samples with undetermined C T value. SD: Standard deviation Table 3: The relationship between the cycle threshold (C T ) and its standard deviation with the concentration of the DNA template when male DNA was targeted (n=30) Standard concentration (ng/μl) kit 1.66-5.56 0.10-0.62 0.006-0.023 0.0016-0.0032 Quantifier Y 5.56 25.8-26.7 26.4 (±1.1) 0.62 29.1-30.1 29.6 (±1.0) 0.023 33.7-37.9 35.8 (±3.1) NA NA NA Quantifiler Duo 5.56 27.3-28.4 27.9 (±1.3) 0.62 30.3-31.9 31.1 (±1.3) 0.023 33.5-37.3* 36.0 (±2.4) NA NA NA Plexor 2.00 21.8-22.7 22.4 (±0.9) 0.40 23.5-25.0 24.4 (±1.6) 0.016 27.3-30.1 29.0 (±2.1) 0.0032 30.2-32.8 31.6 (±2.0) Quantiplex 1.66 25.2-25.8 25.5 (±0.6) 0.10 28.8-29.7 29.3 (±0.7) 0.006 32.5-34.0 33.1 (±1.1) 0.0016 34.1-36.6 35.2 (±2.0) NA indicates DNA quantification is not applicable for Quantifiler Y and Quantifiler Duo in the range of 0.0016-0.0032 ng/μl. *Indicates the detection of samples with undetermined C T value. SD: Standard deviation Preparation of Simulated Touch DNA Samples and STR DNA Typing Analysis A total of 332 simulated touch DNA samples, which were prepared as described previously [19], were used in this study. The extraction of DNA from these touch samples were performed with Chelex 100, Promega DNA IQ TM system, QIAGEN QIAamp DNA Investigator Kit, and QIAGEN QIAsymphony DNA Investigator Kit, respectively, as described [19]. Extracts were then amplified with AmpFlSTR Identifiler PCR Amplification Kit on a Applied Biosystems GeneAmp PCR System 9700 (Life Technologies TM, Carlsbad, CA, USA) and analyzed on the ABI PRISM 3100 Genetic Analyzer (Life Technologies TM, Carlsbad, CA, USA), and Genetic Analyzer, as described [19]. Real-time qpcr Methods and Statistical Analysis DNA quantification of DNA standard and whole blood samples were performed using the Applied Biosystems Quantifiler, Quantifiler Y, Quantifiler Duo Human DNA Kits (Life Technologies TM, Carlsbad, CA, USA), Promega Plexor HY System (Promega, Madison, WI, USA), and QIAGEN Investigator Quantiplex HYres Kit (QIAGEN, Hilden, Germany) in the ABI PRISM 7500 Sequence Detection System (Life Technologies TM, Carlsbad, CA, USA) according to the manufacturer s recommendations. DNA quantification of simulated touch DNA samples were performed using the Applied Biosystems Quantifiler Kit and QIAGEN Investigator Quantiplex HYres Kit in the ABI PRISM 7500 Sequence Detection System. The collected data were analyzed by Applied Biosystems SDS Software v1.0 (Life Technologies TM, Carlsbad, CA, USA). For the data obtained from Plexor, data analysis was accomplished by exporting the collected data to the Plexor Analysis Software. The variation in the concentration for the DNA standards and the whole blood DNA samples were obtained from various quantification methods, and the variation in the average number of loci obtained from the STR analysis were represented as standard deviation (±SD). RESULTS Quantiplex Demonstrates a Higher Precision than Quantifiler, Quantifiler Y, Quantifiler Duo, and Plexor for the of both Autosomal and Male DNA Similar to most PCR-based analysis, real-time PCR quantification is subjected to the stochastic or random sampling effects [20]. When a limited number of DNA target molecules are present in the amplification reaction, the PCR primers in excess will not be able to target the specific DNA regions for quantification, and therefore affecting the precision of the quantification results. In an attempt to compare the precision of the selected quantification kits, we first used their respective DNA standards supplied in the kit and diluted them to the manufacturer s recommended concentration in the following ranges: 2.00-5.56, 0.31-0.62, and 0.016-0.023 ng/μl for autosomal DNA [Table 2], and 1.66-5.56, 0.10-0.62, and 0.006-0.023 ng/μl for male DNA [Table 3]. Regarding the nature of these DNA standards, we believed the results could provide us a clear picture on the precision of each individual quantification kit. The DNA standards for Plexor and Quantiplex were further diluted to 0.0032-0.0049 and 0.0016-0.0032 ng/μl for autosomal Eur J Forensic Sci Apr-Jun 2016 Vol 3 Issue 2 63

and male DNA, respectively, according to manufacturer s recommendations. The diluted samples prepared were then used for DNA quantification, and the cycle threshold, C T values were recorded. Our results demonstrated that C T deviation obtained from Quantiplex was kept below 0.9% for 2.00-5.56, 0.31-0.62, and 0.016-0.023 ng/μl [Table 2]. For the other three kits, the deviations were not varied that much between 2.00-5.56 and 0.31-0.62 ng/μl, and these were kept within 0.6-1.2%, 1.0-1.3%, and 2.1-2.2% for Quantifiler, Quantifiler Duo, and Plexor, respectively, indicating these kits offer a fair consistency in these ranges and were not affected much by stochastic amplification. However, the deviation obtained from these three kits reached a maximum of 1.9%, 2.8%, and 3.0%, respectively, as the DNA concentration dropped to 0.016-0.023 ng/μl, indicating that these kits were subjected to stochastic effect at this range. The above data shows that Quantiplex offered the highest precision of quantification for autosomal DNA with concentration down to 0.016-0.023 ng/μl. We further tested the precisions of both Plexor and Quantiplex at the range of 0.0032-0.0049 ng/μl, i.e. their lowest quantification standards. Our results showed that both Plexor and Quantiplex were affected by stochastic amplification at such low concentration and offered comparable deviations of 3.2% and 3.4%, respectively. When male DNA was targeted, we observed a similar trend of variation in precision from Quantifiler Y, Quantifiler Duo, Plexor, and Quantiplex [Table 3]. Again, Quantiplex offered the highest precision among these kits for the quantification of male DNA, and its deviation was kept below 1.1% even when 0.006-0.023 ng/μl of male DNA was tested as compared to 3.1%, 2.4%, and 2.1% for Quantifiler Y, Quantifiler Duo, and Plexor, respectively. To show the variation of the actual quantification values obtained from these kits, the C T values obtained were converted to concentration by comparing them to their standard curves. The standard curves prepared from each kit were consistent with their respective QC parameters (data not shown). For autosomal DNA, Quantiplex offered a deviation of 9% for both 2.00-5.56 and 0.31-0.62 ng/μl, i.e. the lowest deviation observed among the four tested kits [Figure 1]. On the other hand, Quantifiler, Quantifiler Duo, and Plexor all offered relatively higher deviations of 10-15%, 17-25%, and 15-36%, respectively. At 0.016-0.023 ng/μl, Quantiplex demonstrated the lowest deviation of 18%, whereas Quantifiler, Quantifiler Duo, and Plexor yielded significantly higher deviations of 41%, 43%, and 54%, respectively. At 0.0032-0.0049 ng/μl, Quantiplex deviation increased to 28%, but it was still significantly lower than that from Plexor, which was jumped up to 52%. For the performance of the quantification of male DNA, Quantiplex and Quantifiler Y offered a deviation of 8% only when 1.66-5.56 and 0.10-0.62 ng/μl of male DNA was targeted as compared to 17-31% and 14-32% for Quantifiler Duo and Plexor, respectively [Figure 2]. At 0.006-0.023 ng/μl, Quantiplex continued to offer the highest precision, with a deviation of 19%, as compared to 56%, 62%, and 47% by Quantifiler Y, Quantifiler Duo, and Plexor, respectively. Collectively, our data showed that Quantiplex exhibits the highest precision for the quantification of both autosomal and male DNA. Blood Matrix has Little or No Effect on the Precision of Quantiplex We further compared the precision of these kits on DNA extracted from the whole blood samples, as it is one of the most commonly encountered biological evidence in serious crime and has a relatively complex matrix as compared to the DNA standards. Quantiplex yielded a deviation of 12% and 22% obtained from the high (0.5 ng/μl) and low (0.025 ng/μl) of autosomal DNA, respectively [Figure 3a]. In contrast, Quantifiler, Quantifiler Duo, and Plexor exhibited a lower precision ( high/low ) with deviations of 30/40%, 26/50%, and 19/47%, respectively. Similar performance were obtained when male DNA was targeted, where Quantifiler Y, Quantifiler Duo, Plexor, and Quantiplex exhibited deviations of 17/52%, 25/54%, 13/21%, and 7/17%, respectively [Figure 3b]. These data indicate that Quantiplex exhibits the highest precision not only for the clean DNA standard samples, but also for the whole blood DNA extracts. Quantiplex is a Better Predictor for the Typing Results of Touch DNA Samples as Compared to Quantifiler We have demonstrated from above that Quantiplex shows the highest precision among these kits for both quantifications of autosomal and male DNA. Here, we further explored the effectiveness of Quantiplex in analyzing DNA obtained from touch evidence in which these samples contribute to the majority of the property crime cases. A total of 332 simulated touch DNA samples were collected as described previously [19]. We compared the capability of Quantiplex in predicting the presence of DNA with that of Quantifiler when applying a cut-off threshold of 0.0049 ng/μl and 0.023 ng/μl, i.e. their respective lowest detection limit with reference to DNA typing analysis. We also examined if a true zero value of quantification as STR cut-off could be established. by Quantifiler revealed 189 positive and 143 negative results from the 332 touch DNA samples (using a cut-off threshold of 0.023 ng/μl) [Figure 4a]. 175 and 7 samples out of the 189 positive samples generated typing results with 11 to 15 and 6 to 10 loci, respectively. The remaining 7 samples yielded profiles with <6 loci, and these were referred as poor positive samples, i.e. samples with a concentration above the cut-off threshold yielded profiles with <6 loci. Among the 143 negative samples, only 70 samples i.e. 22 and 48 samples yielded profiles with <6 loci were true negative samples, which contribute to around 49% within the negative samples. The remaining 46 and 27 samples yielded DNA profiles with 6 or more loci were indeed false negative samples, which contributed to 51% of the negative samples. In contrast, Quantiplex yielded 282 positive and 50 negative results (using a cut-off threshold of 0.0049 ng/μl) [Figure 4b]. Among these 282 positive samples, 221 and 30 samples yielded profiles with 11-15 and 6-10 loci, respectively, whereas the remaining 31 samples generated profiles with <6 loci. Among the 50 negative samples, 46 samples (19 and 27 samples with <6 loci) were true negative samples (~92%), whereas only 4 samples were false 64 Eur J Forensic Sci Apr-Jun 2016 Vol 3 Issue 2

Figure 1: Comparison of the precision of quantifi cation kits when autosomal DNA was targeted in their respective DNA standard samples (n = 30). Numerical values (in %) indicated the standard deviations obtained. The rectangular box shows median, upper, and lower quarters of the quantifi cation values. The error bar shows the maximum and minimum quantifi cation values in the particular quantifi cation method. *represents the lowest deviation obtained for that specifi c range of concentration Figure 2: Comparison of the precision of quantification kits when male DNA was targeted in their respective DNA standard samples (n = 30). Numerical values (in %) indicated the standard deviations obtained. The rectangular box shows median, upper, and lower quarters of the quantifi cation values. The error bar shows the maximum and minimum quantifi cation values in the particular quantifi cation method. *represents the lowest deviation obtained for that specifi c range of concentration negative (~8%). In summary, these findings indicate that Quantiplex (true negative: 92%) is a better predictor of DNA typing results than Quantifiler (true negative: ~49%), even though at the cost of yielding nearly three times (11% instead of 4%) more poor positive samples. To further evaluate the sensitivity of Quantiplex, the relationship between the DNA concentration of the tested samples and the number of loci obtained from Identifiler were examined [Table 4]. Our results showed that the average number of loci obtained increased with the quantification values for both Quantifiler and Quantiplex. The key difference between them lies in the effective quantification ranges they offered and the cut-off thresholds we applied. Importantly, Quantiplex with the cut-off threshold set at 0.0049 ng/μl rescued more samples with positive typing results, i.e. a total of 282 samples yielded positive results from at least an average of 9.9 loci and missed only 50 samples (~15%) with an average of 2.0 ± 2.4 loci with limited forensic value. On the other hand, Quantifiler missed a total of 143 samples (~43%) with positive typing results in an average of 6.6 ± 5.1 loci. All these data indicated that Quantiplex is a better predictor of DNA typing results, and hence allowing a better judgment on the subsequent processing of samples. Eur J Forensic Sci Apr-Jun 2016 Vol 3 Issue 2 65

a b Table 4: The relationship of the average number of loci with the quantification value obtained by (a) Quantifiler or (b) Quantiplex for the 332 simulated touch DNA samples value (ng/μl) Number of samples Average number of loci with results (±SD) Quantifier <0.023 143 6.6 (±5.1) (False ve: 73) 0.023-0.050 68 13.3 (±3.2) 0.05 121 14.3 (±2.5) Quantiplex <0.0049 50 2.0 (±2.4) (False +ve: 4) 0.0049-0.022 130 9.9 (±4.4) 0.023-0.050 66 14.1 (±2.8) 0.05 86 14.6 (±2.0) The standard deviation is shown as ±SD. Bold indicates the cut-off threshold of Quantifiler (0.023 ng/μl) and Quantiplex (0.0049 ng/μl), respectively. False -ve indicates samples with concentration below the cut-off threshold yielded profiles with 6 or more loci Figure 3: Comparison of the precision of the quantifi cation kits when (a) Autosomal DNA, or (b) male DNA, was targeted in a whole blood sample (n = 30). Numerical values (in %) shows the standard deviations obtained. The rectangular box shows median, upper, and lower quarters of the quantifi cation values. The error bar shows the maximum and minimum quantifi cation values in the particular quantifi cation method. *represents the lowest deviation obtained for that specifi c range of concentration a b Figure 4: Distribution of DNA profi ling results for the 332 simulated touch DNA samples when quantified by, (a) Quantifiler, and (b) Quantiplex with cut-off threshold at 0.023 ng/μl and 0.0049 ng/μl, respectively. Poor +ve indicates samples with concentration above the cut-off threshold yielded profi les with <6 loci. True ve and false ve indicate samples with concentration below the cut-off threshold yielded profi les with <6 loci and profi les with 6 or more loci, respectively DISCUSSION In the past decade, a number of studies have been conducted on the performance of these qpcr kits. It has been reported that the single copy Quantifiler Duo approach provides better accuracy, whereas the multi-copy Plexor approach offers a higher sensitivity [21], and the authors suggested the selection of kits depends upon the objective of the user. Thomas et al. reported in another study, using a tested half-volume reaction on buccal swabs, that Quantiplex may be useful for predicting STR success of which 66% of the undetected samples yielded no profile [12]. The findings were comparable with previously reported data on Quantifiler of which 73% of the undetected buccal swab samples yielded no profile [5]. Recently, Fregeau and Laurin reported that Quantiplex offers an enhanced performance over the Quantifiler Duo, a newer version of Quantifiler in terms of sensitivity, accuracy, consistency, reliability, and robustness [17]. In this work, in an attempt to obtain the precision of these qpcr kits, we made use of their respective DNA standard as quantification substrates and took advantage of their clean nature, instead of using buccal swabs as reported in other studies [5,12]. Our results demonstrated that Quantiplex displayed a higher precision of quantitation not only for its clean DNA standards, but also for DNA obtained from the complex blood extracts as compared to the other four kits. It is noteworthy that at the range of 0.016-0.023 ng/μl autosomal DNA, Quantiplex offered a precision of ±18%, which is approximately 2-3 times better than those offered by the Quantifiler series (±41-43%) and Plexor (±54%), respectively. This high consistency results generated by Quantiplex, hence, can be used as an effective screening tool for subsequent profiling. Of note, Quantiplex is indeed affected by the stochastic amplification at a very low DNA concentration, i.e. ~10-fold lower as compared to the others. This observation highlights the fact that even quantification kits targeting multi-copy loci are subjected to a certain degree of variability, especially when the target DNA is close to its lower end of quantification. Using a total of 332 simulated touch samples, we were unable to establish a zero cut-off threshold using Quantiplex; however, it improves the consistency and sensitivity of DNA quantification, allowing a better judgment on the subsequent processing of samples. Most of the samples (92%) considered as negative using Quantiplex yielded profiles with limited forensic value. In contrast, only half of them (49%) were true negative using Quantifiler. Considering that Quantifiler (i.e. 62 bp) targets a relatively smaller amplicon than that of the downstream Identifiler STR amplification kit (i.e. 100-450 bp), we expected that Quantifiler will have yielded a greater number 66 Eur J Forensic Sci Apr-Jun 2016 Vol 3 Issue 2

of positive quantification results with poor quality than that of the Quantiplex, which produces a similar amplicon size as compared to the Identifiler (i.e. 146 bp). However, this was not the case as we observed only 7 (~4% of total positive samples) positive samples with poor quality from Quantifiler as compared to 31 (~11% of total positive samples) from that of the Quantiplex. Importantly, Quantiplex also yielded less false negative quantification results than that of the Quantifiler i.e. 4 (~8%) instead of 73 (~51%). This observation could be reconciled by the lower detection threshold limit in Quantiplex, which recovers more samples with low DNA concentration for amplification. To this, one may consider to lower the detection threshold limit of Quantifiler to the range of 0.0049 ng/μl as compared to the Quantiplex. However, our work [Figure 1 and Table 2] demonstrates that in such condition, Quantifiler generates a greater deviation of at least 41% as compared to 28% from Quantiplex. This relatively high inconsistency could affect the effectiveness of Quantifiler as a screening tool. Our study also shows that quantification of the same set of samples by these kits yield different values (data not shown), and such variation could be due to the differences of the copy number of target amplicons and the concentration of their DNA standards [21]. Hence, we strongly recommend that performing a complete validation for both DNA quantification and STR amplification when there is any upgrade of either system, as a new optimal range of input DNA has to be determined for the profiling. 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