- Email: [email protected]
Internal validation of the QIAamp DNA Investigator Kit, QIAamp 96 DNA Swab BioRobot Kit and the BioRobot Universal System for DNA extraction from reference and crime scene samples
G Model
FSIGEN-1259; No. of Pages 3 Forensic Science International: Genetics xxx (2014) e1–e3
Contents lists available at ScienceDirect
Forensic Science International: Genetics journal homepage: www.elsevier.com/locate/fsig
Letter to the Editor Internal validation of the QIAamp DNA Investigator Kit, QIAamp 96 DNA Swab BioRobot Kit and the BioRobot Universal System for DNA extraction from reference and crime scene samples Dear Editor, The quality and quantity of DNA extracted from any forensic sample is of paramount importance for the downstream forensic analyses and ultimately in the resolution of a crime. The extraction procedure can therefore be considered as one of the most critical steps in the molecular manipulations to which evidentiary material is exposed as it is imperative that the integrity and identity of the sample is not jeopardised. The turnover time for laboratory procedures is also of importance for a case so as to meet legal time constraints and ultimately confine perpetrators of crime. To support this endeavour, a combination of forensic genetics armamentaria [i.e., high-throughput platforms for liquid handling and capillary electrophoresis, highly informative and sensitive multiplex human identification systems, laboratory information management systems (LIMS) etc.] cooperatively optimise the workflow and submission of case reports to the justice system within acceptable timeframes to expedite downstream police investigations [1,2]. The present letter summarises the internal validation of the QIAamp DNA Investigator and QIAamp 96 DNA Swab BioRobot Kits for DNA extraction and the BioRobot Universal System an automated liquid handling platform developed by QIAGEN (Hilden, Germany) for forensic applications [3–5]. The necessary experiments were performed in accordance with validation guidelines, quality assurance standards and manufacturer published methods to demonstrate that these commercially available DNA extraction methods perform as expected when they are implemented in our laboratory to extract DNA from both reference (high yield DNA) and crime scene (low yield DNA) samples [3–7]. The validation parameters assessed in this study included accuracy, repeatability, sensitivity, contamination prevention, sample and data tracking, suitability of methods for sample diversity and a comparison of the manual and automated methodologies with respect to DNA extraction efficiency. Biological samples (blood and saliva) were kindly donated with informed consent by laboratory staff for the purpose of this validation study after approval was obtained from the Cyprus National Bioethics Committee for collection and use of these samples in this and other validation studies pertinent to forensic genetic methods. Five donors contributed to the study and each sample was assessed in triplicate per experimental condition (i.e., per volume/per method). The following samples were prepared: cotton swabs with 2, 5, 8 or 10 ml blood; 50 ml saliva deposited on Whatman Omni buccal swabs; small volumes of blood 5–100 ml in tubes, mock crime scene samples including
surface swabs, known non-probative samples (sperm swabs) as well as reference samples (buccal swabs) and finally, National Institute of Standards & Technology (NIST) DNA Profiling Standard SRM 2391c. Mock crime scene validation samples were prepared by in-house sampling so as to represent the spectrum of crime samples commonly encountered in routine analysis. Supplement Table 1 summarises the series of DNA extraction experiments performed to validate both manual and automated methods for both high and low yield samples on dedicated BioRobot Universal instruments. The BioRobot Universal instruments were validated prior to the implementation of the automated DNA extraction methods for routine sample analysis with regard to elution volume accuracy, cross-contamination, sample and data tracking. The volume specified for elution is 75 ml and 125 ml for Test Method 7.2 and Test Method 7.1 respectively. Elution volumes between 40 and 75 ml for Test Method 7.2 have been used successfully. (Supplement Table 2). No cross contamination was observed, all quality controls gave the expected results, all samples and data were easily tracked. Finally, all NIST SRMs extracted and typed gave the profiles indicated in the certificate of analysis [8]. Manual (Test Method 3.1 – Reference Samples; Test Method 3.2 – Crime Scene samples) and automated (Test Method 7.1 – Reference Samples; Test Method 7.2 – Crime Scene samples) methods were implemented according to manufacturer instructions [3–5]. DNA yield and purity were assessed by Real Time PCR using a modified published method running on the Applied Biosystems 7500 Real Time PCR System with the Sequence Detection Software (SDS) v 1.2.3 [9]. The quality of DNA isolated was assessed by autosomal STR PCR analysis using the PowerPlex 16 STR System from Promega in accordance with manufacturer instructions [10]. The analytical threshold for this validation study was set at 50 RFU. The profiles were classified as full or partial and number of amplified loci and relative fluorescent units (RFU) were recorded. Where sufficient DNA was isolated, 1 ng of template was used for STR PCR. If <1 ng was available, then maximum template volume (19.2 ml) was used for STR PCR set-up. It was observed that for reference DNA extraction methods, the automated method, Test Method 7.1 gave lower DNA yields when compared to the manual method, Test Method 3.1. The DNA was of sufficient quantity and good quality for STR analysis as exemplified by the STR data. These lower yields, served to reduce the dilution factor required for downstream sample analysis and thus facilitate the liquid handling steps of the BioRobots. The manual method, Test Method 3.1 gave higher yields which require further dilution which is easier to achieve manually. (Supplement Tables 3–4). For the test methods designed to extract DNA from crime scene samples (Test Method 3.2 and Test Method 7.2) it is noted that for the extraction of blood from cotton swabs, 12/20 samples gave
http://dx.doi.org/10.1016/j.fsigen.2014.10.020 1872-4973/ß 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: S. Xenophontos, et al., Internal validation of the QIAamp DNA Investigator Kit, QIAamp 96 DNA Swab BioRobot Kit and the BioRobot Universal System for DNA extraction from reference and crime scene samples, Forensic Sci. Int. Genet. (2014), http://dx.doi.org/10.1016/j.fsigen.2014.10.020
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FSIGEN-1259; No. of Pages 3 e2
Letter to the Editor / Forensic Science International: Genetics xxx (2014) e1–e3
higher yields when extracted by the automated method as opposed to the manual method, similarly for the small volumes of blood in tubes, 13 of 20 samples gave higher yields when extracted via the automated method as opposed to the manual method (Supplement Tables 5–6). Taking into consideration the standard deviations we observe that both are equally efficient in extracting DNA yields of sufficient quality and quantity for STR analysis. As the same chemistry is used for extraction purposes any variation in the extraction efficiency as determined by the DNA concentration can be accounted for by any or a combination of factors such as the number of white blood cells in the volumes aliquoted in the tubes or deposited on swabs for extraction, pipetting error at this stage and any other stage during the manual procedure, variation in the incubation times or elution efficiency through the QIAamp columns or even in the quantitation method. For the same reasons, the repeatability of the extraction methods was difficult to demonstrate for the cotton swab blood deposits for volumes of 10, 8, 5 and 2 ml which were also too close to distinguish differences between each volume. The repeatability of the extraction methods (Test Method 3.2 and Test Method 7.2) was best observed for the small volumes of blood extracted using volumes of 40, 20, 10 and 5 ml aliquoted in tubes, which not only indicated comparatively small values in the standard deviations of the triplicate means but the DNA yields/DNA concentrations also conformed to the expected ratio of starting material (Supplement Tables 7–8). The coefficient of variation indicates an increasing variability between manual and automated DNA extraction methods as the starting blood volume decreases (Supplement Table 9). Since both methods will be employed for routine work it is acceptable that both yield sufficient DNA of good quality for the downstream analysis. These observations are in agreement with previous studies which compared manual and automated methods based on one of QIAGEN’s BioRobot Universal System predecessors; the QIAcube liquid handling system and QIAamp Investigator chemistry which showed that yields were comparable in both methods [11,12]. In contrast, the comparative analysis based on another BioRobot Universal System predecessor; BioRobot EZ1 did not culminate in the same performance parameters but instead demonstrated the ‘‘underperformance’’ of BioRobot EZ1 compared to both QIAcube and manual extraction procedure [12]. The BioRobot Universal has been shown to perform as efficiently as manual methods in our validation study in the isolation of DNA from both low and high yield DNA bearing samples. From the successive reductions in volumes of blood deposited on cotton swabs for extraction via Test Method 3.2 and Test Method 7.2, it was observed that successful extraction was achieved from as low as 2 ml volumes. The Mock crime scene sample DNA extraction experiment exposed Test Method 7.2 to the most stringent validation as here simulated case samples with trace DNA gave full STR profiles for 72% of samples (Supplement Table 10). It was observed in some instances that even though a zero reading was obtained from quantitation data, a full STR profile was obtained upon amplification using full template volume (19.2 ml) for the STR PCR. Such observations have served to improve the guidelines of the lab for STR PCR set up in the context of low or zero quantitation data as significant profiles may be lost if caution is not exercised. Similar inconsistencies between DNA quantitation and STR data have been reported previously [13]. The overall results accumulated from this DNA extraction validation study based on QIAGEN’s QIAamp DNA Investigator chemistry demonstrate the reliability, suitability and robustness of all methods. From a total of 326 STR amplifications from the various DNA samples extracted, a first pass full profile success was achieved for 304/326 reactions (93% of the total). If one considers the 7% of samples that did not give a full profile which arise from the mock crime scene samples, it is noted that 18 of these gave
profiles which had 10–15 loci. Inclusion of these samples in the group of first pass full profile reactions gives a total of 322 exploitable profiles or a total of 99%. This is obviously an acceptable result to demonstrate that the DNA extraction methods on all types of samples provide satisfactory DNA yields of acceptable quality for downstream STR analyses. No inhibition was observed in the reactions as exemplified by the IPC data of quantitation experiments and the STR experiments illustrating that all inhibitors of molecular analyses such as haem from blood samples had been successfully removed. The most critical objective of the present validation study was to validate the BioRobot Universal System and QIAGEN’s Investigator Chemistry as a new automated DNA extraction method for crime scene samples which are usually compromised and may contain only trace amounts of DNA which are frequently complex mixtures. The evaluation of STR profiles obtained from the variety of mock crime scene samples indicates that in 72% of samples (56/78), a full STR profile was obtained. In addition, where the biological material deposited on the sample or surface swab was of known origin, the profile(s) obtained was/were that/those expected from the donor(s) and only a small number showed allele or locus drop out which is accounted for by the 28% or 22 samples and amongst these only 2 failed to amplify at all loci. If we consider that investigative information can be obtained from samples with at least 10 amplified loci, then in 95% of mock crime scene samples (74/78) a first pass success rate was achieved. From the cumulative data, illustrated in Supplemental Table 11 and representative electropherograms in Figs. 1–3, it is concluded that via this method (Test Method 7.2) running on the BioRobot Universal System, good quality DNA has been retrieved to give quality profiles. We fully understand and expect that real case samples, exposed to various environmental insults, may behave differently from our mock crime scene samples in terms of the ability to extract DNA successfully and/or obtain full DNA profiles from them. In light of all the aforementioned data with regard to the set validation criteria, all the DNA extraction methods are deemed acceptable for implementation in the laboratory for routine case work. Acknowledgements The authors wish to thank the laboratory colleagues for donating samples for this study. Funding was provided by the European Regional Development Fund and the Republic of Cyprus through the Research Promotion Foundation (Project NEA YPODOMH/NEKYP/0308/12). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.fsigen.2014.10.020. References [1] B. Budowle, A. Van Daal, Extracting evidence from forensic DNA analyses: future molecular biology directions, BioTechniques 46 (5) (2009) 339–350. [2] B. Keating, A.T. Bansal, S. Walsh, J. Millman, J. Newman, B. Budowle, A. Eisenberg, J. Donfack, P. Gasparini, Z. Budimlija, A.K. Henders, H. Chandrupatla, D.L. Duffy, S.D. Gordon, P. Hysi, F. Liu, S.E. Medland, L. Rubin, N.G. Martin, T.D. Spector, M. Kayser, International Visible Trait Genetics (VisiGen) Consortium, First all-in-one diagnostic tool for DNA intelligence: genome-wide inference of biogeographic ancestry, appearance, relatedness, and sex with Identitas v1 Forensic Chip, Int. J. Legal Med. 127 (3) (2013) 559–572. [3] QIAamp DNA Investigator Handbook, April 2010. [4] QIAamp 96 DNA Swab BioRobot Kit Handbook, April 2010. [5] BioRobot Universal System User Manual Version 1.0, January 2007. [6] General requirements for the competence of testing and calibration laboratories – EN ISO/IEC 17025, 2005.
Please cite this article in press as: S. Xenophontos, et al., Internal validation of the QIAamp DNA Investigator Kit, QIAamp 96 DNA Swab BioRobot Kit and the BioRobot Universal System for DNA extraction from reference and crime scene samples, Forensic Sci. Int. Genet. (2014), http://dx.doi.org/10.1016/j.fsigen.2014.10.020
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FSIGEN-1259; No. of Pages 3 Letter to the Editor / Forensic Science International: Genetics xxx (2014) e1–e3 [7] Scientific Working Group on DNA Analysis Methods – Validation Guidelines for DNA Analysis Methods, 2012. [8] National Institute of Standards & Technology Certificate of Analysis Standard Reference Material 2391, PCR-Based DNA Profiling Standard, Certificate Issue Date: 11 August 201. [9] K.L. Swango, W.R. Hudlow, M.D. Timken, M.R. Buoncristiani, Developmental validation of a multiplex qPCR assay for assessing the quantity and quality of nuclear DNA in forensic samples, Forensic Sci. Int. 170 (1) (2007) 35–45. [10] Technical Manual PowerPlex 16 System–Instructions for use of Products (DC6530 and DC6531). [11] K. Phillips, N. McCallum, L. Welch, A comparison of methods for forensic DNA extraction: Chelex-100 and the QIAGEN DNA Investigator Kit (manual and automated), Forensic Sci. Int. Genet. 6 (2) (2012) 282–285. [12] R.J. Brownlow, K.E. Dagnall, C.E. Ames, A comparison of DNA collection and retrieval from two swab types (cotton and nylon flocked swab) when processed using three QIAGEN extraction methods, J. Forensic Sci. 57 (3) (2012) 713–717. [13] C. Rucinski, A.L. Malaver, E.J. Yunis, J.J. Yunis, Comparison of two methods for isolating DNA from human skeletal remains for STR analysis, J. Forensic Sci. 57 (3) (2012) 706–712.
e3
Stavroulla Xenophontos Vasilis Christofi George Iosif Pavlos Polycarpou Panayiotis Manoli Nafsika Demetriou Marios A. Cariolou* The Cyprus Institute of Neurology & Genetics, Laboratory of Forensic Genetics, 6 Airport Avenue, 2370 Nicosia, Cyprus *Corresponding
author. Tel.: +357 22 392651; fax: +357 22 392638 E-mail address: [email protected] (M.A. Cariolou). Received 22 August 2014
Revised 15 October 2014 Accepted 24 October 2014
Please cite this article in press as: S. Xenophontos, et al., Internal validation of the QIAamp DNA Investigator Kit, QIAamp 96 DNA Swab BioRobot Kit and the BioRobot Universal System for DNA extraction from reference and crime scene samples, Forensic Sci. Int. Genet. (2014), http://dx.doi.org/10.1016/j.fsigen.2014.10.020
FSIGEN-1259; No. of Pages 3 Forensic Science International: Genetics xxx (2014) e1–e3
Contents lists available at ScienceDirect
Forensic Science International: Genetics journal homepage: www.elsevier.com/locate/fsig
Letter to the Editor Internal validation of the QIAamp DNA Investigator Kit, QIAamp 96 DNA Swab BioRobot Kit and the BioRobot Universal System for DNA extraction from reference and crime scene samples Dear Editor, The quality and quantity of DNA extracted from any forensic sample is of paramount importance for the downstream forensic analyses and ultimately in the resolution of a crime. The extraction procedure can therefore be considered as one of the most critical steps in the molecular manipulations to which evidentiary material is exposed as it is imperative that the integrity and identity of the sample is not jeopardised. The turnover time for laboratory procedures is also of importance for a case so as to meet legal time constraints and ultimately confine perpetrators of crime. To support this endeavour, a combination of forensic genetics armamentaria [i.e., high-throughput platforms for liquid handling and capillary electrophoresis, highly informative and sensitive multiplex human identification systems, laboratory information management systems (LIMS) etc.] cooperatively optimise the workflow and submission of case reports to the justice system within acceptable timeframes to expedite downstream police investigations [1,2]. The present letter summarises the internal validation of the QIAamp DNA Investigator and QIAamp 96 DNA Swab BioRobot Kits for DNA extraction and the BioRobot Universal System an automated liquid handling platform developed by QIAGEN (Hilden, Germany) for forensic applications [3–5]. The necessary experiments were performed in accordance with validation guidelines, quality assurance standards and manufacturer published methods to demonstrate that these commercially available DNA extraction methods perform as expected when they are implemented in our laboratory to extract DNA from both reference (high yield DNA) and crime scene (low yield DNA) samples [3–7]. The validation parameters assessed in this study included accuracy, repeatability, sensitivity, contamination prevention, sample and data tracking, suitability of methods for sample diversity and a comparison of the manual and automated methodologies with respect to DNA extraction efficiency. Biological samples (blood and saliva) were kindly donated with informed consent by laboratory staff for the purpose of this validation study after approval was obtained from the Cyprus National Bioethics Committee for collection and use of these samples in this and other validation studies pertinent to forensic genetic methods. Five donors contributed to the study and each sample was assessed in triplicate per experimental condition (i.e., per volume/per method). The following samples were prepared: cotton swabs with 2, 5, 8 or 10 ml blood; 50 ml saliva deposited on Whatman Omni buccal swabs; small volumes of blood 5–100 ml in tubes, mock crime scene samples including
surface swabs, known non-probative samples (sperm swabs) as well as reference samples (buccal swabs) and finally, National Institute of Standards & Technology (NIST) DNA Profiling Standard SRM 2391c. Mock crime scene validation samples were prepared by in-house sampling so as to represent the spectrum of crime samples commonly encountered in routine analysis. Supplement Table 1 summarises the series of DNA extraction experiments performed to validate both manual and automated methods for both high and low yield samples on dedicated BioRobot Universal instruments. The BioRobot Universal instruments were validated prior to the implementation of the automated DNA extraction methods for routine sample analysis with regard to elution volume accuracy, cross-contamination, sample and data tracking. The volume specified for elution is 75 ml and 125 ml for Test Method 7.2 and Test Method 7.1 respectively. Elution volumes between 40 and 75 ml for Test Method 7.2 have been used successfully. (Supplement Table 2). No cross contamination was observed, all quality controls gave the expected results, all samples and data were easily tracked. Finally, all NIST SRMs extracted and typed gave the profiles indicated in the certificate of analysis [8]. Manual (Test Method 3.1 – Reference Samples; Test Method 3.2 – Crime Scene samples) and automated (Test Method 7.1 – Reference Samples; Test Method 7.2 – Crime Scene samples) methods were implemented according to manufacturer instructions [3–5]. DNA yield and purity were assessed by Real Time PCR using a modified published method running on the Applied Biosystems 7500 Real Time PCR System with the Sequence Detection Software (SDS) v 1.2.3 [9]. The quality of DNA isolated was assessed by autosomal STR PCR analysis using the PowerPlex 16 STR System from Promega in accordance with manufacturer instructions [10]. The analytical threshold for this validation study was set at 50 RFU. The profiles were classified as full or partial and number of amplified loci and relative fluorescent units (RFU) were recorded. Where sufficient DNA was isolated, 1 ng of template was used for STR PCR. If <1 ng was available, then maximum template volume (19.2 ml) was used for STR PCR set-up. It was observed that for reference DNA extraction methods, the automated method, Test Method 7.1 gave lower DNA yields when compared to the manual method, Test Method 3.1. The DNA was of sufficient quantity and good quality for STR analysis as exemplified by the STR data. These lower yields, served to reduce the dilution factor required for downstream sample analysis and thus facilitate the liquid handling steps of the BioRobots. The manual method, Test Method 3.1 gave higher yields which require further dilution which is easier to achieve manually. (Supplement Tables 3–4). For the test methods designed to extract DNA from crime scene samples (Test Method 3.2 and Test Method 7.2) it is noted that for the extraction of blood from cotton swabs, 12/20 samples gave
http://dx.doi.org/10.1016/j.fsigen.2014.10.020 1872-4973/ß 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: S. Xenophontos, et al., Internal validation of the QIAamp DNA Investigator Kit, QIAamp 96 DNA Swab BioRobot Kit and the BioRobot Universal System for DNA extraction from reference and crime scene samples, Forensic Sci. Int. Genet. (2014), http://dx.doi.org/10.1016/j.fsigen.2014.10.020
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FSIGEN-1259; No. of Pages 3 e2
Letter to the Editor / Forensic Science International: Genetics xxx (2014) e1–e3
higher yields when extracted by the automated method as opposed to the manual method, similarly for the small volumes of blood in tubes, 13 of 20 samples gave higher yields when extracted via the automated method as opposed to the manual method (Supplement Tables 5–6). Taking into consideration the standard deviations we observe that both are equally efficient in extracting DNA yields of sufficient quality and quantity for STR analysis. As the same chemistry is used for extraction purposes any variation in the extraction efficiency as determined by the DNA concentration can be accounted for by any or a combination of factors such as the number of white blood cells in the volumes aliquoted in the tubes or deposited on swabs for extraction, pipetting error at this stage and any other stage during the manual procedure, variation in the incubation times or elution efficiency through the QIAamp columns or even in the quantitation method. For the same reasons, the repeatability of the extraction methods was difficult to demonstrate for the cotton swab blood deposits for volumes of 10, 8, 5 and 2 ml which were also too close to distinguish differences between each volume. The repeatability of the extraction methods (Test Method 3.2 and Test Method 7.2) was best observed for the small volumes of blood extracted using volumes of 40, 20, 10 and 5 ml aliquoted in tubes, which not only indicated comparatively small values in the standard deviations of the triplicate means but the DNA yields/DNA concentrations also conformed to the expected ratio of starting material (Supplement Tables 7–8). The coefficient of variation indicates an increasing variability between manual and automated DNA extraction methods as the starting blood volume decreases (Supplement Table 9). Since both methods will be employed for routine work it is acceptable that both yield sufficient DNA of good quality for the downstream analysis. These observations are in agreement with previous studies which compared manual and automated methods based on one of QIAGEN’s BioRobot Universal System predecessors; the QIAcube liquid handling system and QIAamp Investigator chemistry which showed that yields were comparable in both methods [11,12]. In contrast, the comparative analysis based on another BioRobot Universal System predecessor; BioRobot EZ1 did not culminate in the same performance parameters but instead demonstrated the ‘‘underperformance’’ of BioRobot EZ1 compared to both QIAcube and manual extraction procedure [12]. The BioRobot Universal has been shown to perform as efficiently as manual methods in our validation study in the isolation of DNA from both low and high yield DNA bearing samples. From the successive reductions in volumes of blood deposited on cotton swabs for extraction via Test Method 3.2 and Test Method 7.2, it was observed that successful extraction was achieved from as low as 2 ml volumes. The Mock crime scene sample DNA extraction experiment exposed Test Method 7.2 to the most stringent validation as here simulated case samples with trace DNA gave full STR profiles for 72% of samples (Supplement Table 10). It was observed in some instances that even though a zero reading was obtained from quantitation data, a full STR profile was obtained upon amplification using full template volume (19.2 ml) for the STR PCR. Such observations have served to improve the guidelines of the lab for STR PCR set up in the context of low or zero quantitation data as significant profiles may be lost if caution is not exercised. Similar inconsistencies between DNA quantitation and STR data have been reported previously [13]. The overall results accumulated from this DNA extraction validation study based on QIAGEN’s QIAamp DNA Investigator chemistry demonstrate the reliability, suitability and robustness of all methods. From a total of 326 STR amplifications from the various DNA samples extracted, a first pass full profile success was achieved for 304/326 reactions (93% of the total). If one considers the 7% of samples that did not give a full profile which arise from the mock crime scene samples, it is noted that 18 of these gave
profiles which had 10–15 loci. Inclusion of these samples in the group of first pass full profile reactions gives a total of 322 exploitable profiles or a total of 99%. This is obviously an acceptable result to demonstrate that the DNA extraction methods on all types of samples provide satisfactory DNA yields of acceptable quality for downstream STR analyses. No inhibition was observed in the reactions as exemplified by the IPC data of quantitation experiments and the STR experiments illustrating that all inhibitors of molecular analyses such as haem from blood samples had been successfully removed. The most critical objective of the present validation study was to validate the BioRobot Universal System and QIAGEN’s Investigator Chemistry as a new automated DNA extraction method for crime scene samples which are usually compromised and may contain only trace amounts of DNA which are frequently complex mixtures. The evaluation of STR profiles obtained from the variety of mock crime scene samples indicates that in 72% of samples (56/78), a full STR profile was obtained. In addition, where the biological material deposited on the sample or surface swab was of known origin, the profile(s) obtained was/were that/those expected from the donor(s) and only a small number showed allele or locus drop out which is accounted for by the 28% or 22 samples and amongst these only 2 failed to amplify at all loci. If we consider that investigative information can be obtained from samples with at least 10 amplified loci, then in 95% of mock crime scene samples (74/78) a first pass success rate was achieved. From the cumulative data, illustrated in Supplemental Table 11 and representative electropherograms in Figs. 1–3, it is concluded that via this method (Test Method 7.2) running on the BioRobot Universal System, good quality DNA has been retrieved to give quality profiles. We fully understand and expect that real case samples, exposed to various environmental insults, may behave differently from our mock crime scene samples in terms of the ability to extract DNA successfully and/or obtain full DNA profiles from them. In light of all the aforementioned data with regard to the set validation criteria, all the DNA extraction methods are deemed acceptable for implementation in the laboratory for routine case work. Acknowledgements The authors wish to thank the laboratory colleagues for donating samples for this study. Funding was provided by the European Regional Development Fund and the Republic of Cyprus through the Research Promotion Foundation (Project NEA YPODOMH/NEKYP/0308/12). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.fsigen.2014.10.020. References [1] B. Budowle, A. Van Daal, Extracting evidence from forensic DNA analyses: future molecular biology directions, BioTechniques 46 (5) (2009) 339–350. [2] B. Keating, A.T. Bansal, S. Walsh, J. Millman, J. Newman, B. Budowle, A. Eisenberg, J. Donfack, P. Gasparini, Z. Budimlija, A.K. Henders, H. Chandrupatla, D.L. Duffy, S.D. Gordon, P. Hysi, F. Liu, S.E. Medland, L. Rubin, N.G. Martin, T.D. Spector, M. Kayser, International Visible Trait Genetics (VisiGen) Consortium, First all-in-one diagnostic tool for DNA intelligence: genome-wide inference of biogeographic ancestry, appearance, relatedness, and sex with Identitas v1 Forensic Chip, Int. J. Legal Med. 127 (3) (2013) 559–572. [3] QIAamp DNA Investigator Handbook, April 2010. [4] QIAamp 96 DNA Swab BioRobot Kit Handbook, April 2010. [5] BioRobot Universal System User Manual Version 1.0, January 2007. [6] General requirements for the competence of testing and calibration laboratories – EN ISO/IEC 17025, 2005.
Please cite this article in press as: S. Xenophontos, et al., Internal validation of the QIAamp DNA Investigator Kit, QIAamp 96 DNA Swab BioRobot Kit and the BioRobot Universal System for DNA extraction from reference and crime scene samples, Forensic Sci. Int. Genet. (2014), http://dx.doi.org/10.1016/j.fsigen.2014.10.020
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FSIGEN-1259; No. of Pages 3 Letter to the Editor / Forensic Science International: Genetics xxx (2014) e1–e3 [7] Scientific Working Group on DNA Analysis Methods – Validation Guidelines for DNA Analysis Methods, 2012. [8] National Institute of Standards & Technology Certificate of Analysis Standard Reference Material 2391, PCR-Based DNA Profiling Standard, Certificate Issue Date: 11 August 201. [9] K.L. Swango, W.R. Hudlow, M.D. Timken, M.R. Buoncristiani, Developmental validation of a multiplex qPCR assay for assessing the quantity and quality of nuclear DNA in forensic samples, Forensic Sci. Int. 170 (1) (2007) 35–45. [10] Technical Manual PowerPlex 16 System–Instructions for use of Products (DC6530 and DC6531). [11] K. Phillips, N. McCallum, L. Welch, A comparison of methods for forensic DNA extraction: Chelex-100 and the QIAGEN DNA Investigator Kit (manual and automated), Forensic Sci. Int. Genet. 6 (2) (2012) 282–285. [12] R.J. Brownlow, K.E. Dagnall, C.E. Ames, A comparison of DNA collection and retrieval from two swab types (cotton and nylon flocked swab) when processed using three QIAGEN extraction methods, J. Forensic Sci. 57 (3) (2012) 713–717. [13] C. Rucinski, A.L. Malaver, E.J. Yunis, J.J. Yunis, Comparison of two methods for isolating DNA from human skeletal remains for STR analysis, J. Forensic Sci. 57 (3) (2012) 706–712.
e3
Stavroulla Xenophontos Vasilis Christofi George Iosif Pavlos Polycarpou Panayiotis Manoli Nafsika Demetriou Marios A. Cariolou* The Cyprus Institute of Neurology & Genetics, Laboratory of Forensic Genetics, 6 Airport Avenue, 2370 Nicosia, Cyprus *Corresponding
author. Tel.: +357 22 392651; fax: +357 22 392638 E-mail address: [email protected] (M.A. Cariolou). Received 22 August 2014
Revised 15 October 2014 Accepted 24 October 2014
Please cite this article in press as: S. Xenophontos, et al., Internal validation of the QIAamp DNA Investigator Kit, QIAamp 96 DNA Swab BioRobot Kit and the BioRobot Universal System for DNA extraction from reference and crime scene samples, Forensic Sci. Int. Genet. (2014), http://dx.doi.org/10.1016/j.fsigen.2014.10.020