Forensic Science International: Genetics

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1 Forensic Science International: Genetics 4 (2009) Contents lists available at ScienceDirect Forensic Science International: Genetics journal homepage: Evaluation of the 124-plex SNP typing microarray for forensic testing Kaarel Krjutškov a,b,c, *, Triin Viltrop a, Priit Palta b,d, Ene Metspalu g, Erika Tamm g, Siim Suvi c, Katrin Sak c, Alo Merilo k, Helena Sork e, Rita Teek f,g, Tiit Nikopensius a, Toomas Kivisild h,i, Andres Metspalu a,b,j a Department of Biotechnology, IMCB, University of Tartu, 23 Riia St, Tartu, Estonia b Estonian Biocentre, 23 Riia St, Tartu, Estonia c Asper Biotech, 17A Vaksali St, Tartu, Estonia d Department of Bioinformatics, IMCB, University of Tartu, 23 Riia St, Tartu, Estonia e Institute of Technology, University of Tartu, 1 Nooruse St, 50411, Tartu, Estonia f Department of Oto-Rhino-Laryngology, University of Tartu, 1 Kuperjanovi St, Tartu, Estonia g Department of Genetics, United Laboratories, Tartu University Hospital, 3 Oru St, Tartu, Estonia h Leverhulme Centre for Human Evolutionary Studies, University of Cambridge, CB2 1QH Cambridge, UK i Department of Evolutionary Biology, IMCB, University of Tartu, 23 Riia St, Tartu, Estonia j Estonian Genome Project of University of Tartu, 61b Tiigi St, Tartu, Estonia k Harcourt Management Consulting, 7 Sonajala St, Tartu, Estonia ARTICLE INFO ABSTRACT Article history: Received 22 December 2008 Received in revised form 16 April 2009 Accepted 17 April 2009 Keywords: Multiplex PCR Forensic test APEX-2 genotyping Human identification systems such as criminal databases, forensic DNA testing and genetic genealogy require reliable and cost-effective genotyping of autosomal, mitochondrial and Y chromosome markers from different biological materials, including venous blood and saliva. Although many such assays are available, few systems are capable of simultaneously detecting all three targets in a single reaction. Employing the APEX-2 principle, we have characterized a novel 124-plex assay, using specific primer extension, universal primer amplification and single base extension on an oligonucleotide array. The assay has been designed for simultaneous genotyping of SNPs from the single copy loci (46 autosomal and 29 Y chromosomal markers) side by side with SNPs from the mitochondrial genome (49 markers) that appears in up to thousands of copies per cell in certain tissue types. All the autosomal SNPs (from the SNPforID Consortium) included in the multiplex assay are unlinked and are distributed widely across autosomes, enabling genetic fingerprints to be distinguished. Mitochondrial DNA and Y chromosome polymorphisms that define haplogroups common in European populations are included to allow for maternity and paternity testing and for the analysis of genetic genealogies. After assay optimization we estimated the accuracy (99.83%) and call rate (99.66%) of the protocol on 17 mother father child/ children families and five internal control DNAs. In addition, 79 unrelated Estonian and Swedish DNA samples were genotyped and the accuracy of mtdna and Y chromosome haplogroup inference by the multiplex method was assessed using conventional genotyping methods and direct sequencing. ß 2009 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Compared to conventional STR-based methods, single nucleotide polymorphisms (SNPs) show increasing potential for forensic case-work studies because a number of features make them attractive: (i) there are several millions of validated SNPs in the human genome; (ii) SNPs can be typed by automated, costeffective and standardized methods; and (iii) PCR amplicons shorter than 100 bp allow for amplification and unambiguous * Corresponding author at: Department of Biotechnology, IMCB, University of Tartu, 23 Riia St., Tartu, Estonia. Tel.: ; fax: address: [email protected] (K. Krjutškov). allele calling even from highly degraded DNA samples [1]. (iv) The mutation rate of SNPs is lower ((2 4) 10 8 /site/generation [2]) than those of STRs or VNTRs (variable number tandem repeats, e.g. 0.23%/STR/generation [3]); and (v) as biallelic polymorphisms, SNPs are comparatively easy to validate [4]. Besides many advantages over STR-based systems, significant disadvantages using SNPs, should also be keep in mind. (i) Mixture analysis is still an obstacle. A major advantage with STRs in a forensic setting is that many possible alleles exist, providing the possibility that the multiple contributors to a mixture will have distinguishable (non-overlapping) alleles [5]. (ii)another reason why SNP technology would not dominate forensic DNA analysis in the near future is that national DNA databases store STR data and SNP profiles cannot be searched against an STR /$ see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi: /j.fsigen

2 44 K. Krjutškov et al. / Forensic Science International: Genetics 4 (2009) profiles; and (iii) SNP multiplex reactions require more target DNA than STR tests (31 pg 2 ng) [6]. The SNPforID Consortium has selected more than 50 autosomal SNPs that are suitable for identifying individuals of unknown population origin and for determining allele frequencies in major populations [4]. The SNPforID study investigated that a total of 52 SNPs reported to be polymorphic in European, Asian and African populations. These polymorphic variants served as a basis for developing a highly sensitive SNP-typing method, which allowed all 52 DNA fragments to be amplified in one PCR reaction and subsequently detected in two single base extension (SBE) reactions by capillary electrophoresis (non-commercial kit). The SNPforID assay was tested on 124 mother child father trios, and three or more mismatches were found in 99.85% of the 83,096 comparisons between mother, child and an unrelated male. Only two of the 83,096 comparisons matched perfectly between an unrelated male and the mother child duo [7]. In addition to this 52-multiplex PCR, a 49-plex [8] and 34 SNP-assay [9] have been developed. Like the 52-multiplex, the 49-marker Genplex typing system (modification of SNPlex TM ) uses SNPforID markers while the 34 SNP set comprises markers that are amplified in four multiplex PCRs and are highly polymorphic particularly in East Asian populations. Human mitochondrial DNA (mtdna) is a 16,569 bp circular molecule present in hundreds or thousands of extranuclear copies per cell. Its abundance makes it possible to obtain high yields of mtdna from a wide range of biological sources except sperm. The maternal inheritance and high mutation rate make mtdna useful for evolutionary and forensic studies [10]. Commercial multiplex PCR kits that are currently available enable 36 haplotype-specific mtsnps to be covered using four 9-plex sets in the East Asian population [11], 12 mtsnps in a 12-plex reaction for anthropological studies [12] and 22 mtsnps (22-plex) [13]. A more sensitive assay (five-genome equivalents of mtdna per reaction) using an 18-plex and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI) for analyzing largescale samples or limited amounts of mtdna was published recently [14]. Analysis of Y chromosome SNPs has proved valuable for both human evolutionary studies and forensic identification purposes. The low mutation rate, paternal inheritance and absence of recombination make Y-SNPs particularly suitable for identifying paternal lineages to reconstruct ancestral states and explore family relationships. Since 2005, many groups have shown that SNP markers can provide useful information for analyzing forensic cases and have developed optimized and very sensitive Y-SNP multiplex genotyping methods [15,16]. A sensitivity test for 13 Y- SNPs, using varying amounts of DNA, displayed high reliability with ng of the template [17]. The positions were analyzed by simultaneous amplification in two separate multiplex PCRs followed by allelic discrimination in one multiplex minisequencing reaction. The aim of the present study was to demonstrate the amplification of 49 mitochondrial, 29 Y chromosomal and 46 autosomal SNP markers simultaneously in a 124-multiplex APEX-2 reaction and to analyze alleles by SBE on the microarrays. To this end we selected identification-informative genetic markers, and we optimized and validated the whole protocol by analyzing a set of 17 families, 79 unrelated samples and five internal controls. 2. Materials and methods 2.1. DNA samples and extraction All blood samples for DNA extraction were collected after obtaining informed written consent from 101 healthy volunteers. Blood (4.5 ml) was collected in lavender-top EDTA BD Vacutainer TM tubes (BD, NY, USA). Saliva samples from 25 volunteers were collected following the manufacturer s instructions and kept for 1 2 days at room temperature before DNA extraction. DNA was isolated from saliva using a PSP Saligene DNA Kit (Invitek GmbH, Berlin, Germany). DNA was extracted from blood using Genomic DNA Purification Kit (Fermentas, Vilnius, Lithuania) with minor modifications: after chloroform had been added, the sample was mixed until the solution was homogenous; all centrifugations were performed at 16,000 g and after the precipitation solution was added the centrifugation lasted for 10 min. Eluation was carried out in 100 ml of eluation solution (5 mm Tris HCl, ph 8.5). The DNA concentration and A 260/280 were measured with a NanoDrop TM 1000 Spectrophotometer (Thermo Scientific, Waltham, USA) Selection of polymorphic loci The initial 132-plex assay included 50 mtdna markers selected from the phylogeny based on complete mtdna sequences as defining markers from the major branches of common haplogroups in Europe, the Near East and North Africa [18 21]. A phylogenetic tree showing the markers included and the respective haplogroup definitions is presented in Supplementary Fig. 1A. To minimize the effect of preponderant homoplasy, the markers were selected primarily from the coding region. The 30 Y chromosome markers were chosen from the Y chromosome haplogroup tree to represent clades common in Europe, the Near East and North Africa [21]. Haplogroup definitions and marker names are shown in Supplementary Fig. 1B and are in accordance with the revised nomenclature of Y Chromosome Consortium [22]. A total of 52 SNPs, reported to be polymorphic in European, Asian and African populations [4], evaluated in a collaborative exercise [23] and paternity testing [7], were provided from the SNPforID Consortium homepage ( The APEX-2 forensic assay The main principle of the APEX-2 forensic assay system is to identify the nucleotide under investigation after one nucleotide 124-plex amplification on an oligoarray using the SBE reaction [24]. The assay comprises the following reaction phases (Fig. 1) performed in an eight-strip-well format: (i) First phase. During PCR with specific primers, hundreds of different positions are amplified simultaneously. Each position requires two oligonucleotides consisting of three parts: specific, universal part and a 5 0 modification. The 3 0 end of the primer lies just before the polymorphism and only one nucleotide (SNP) is unknown in the whole amplicon ( bp; specific regions without universal primer sequences are in the range bp (Fig. 1D)). (ii) Second phase. The reaction volume is increased 30 times (150 ml) to guarantee sufficient material for array-format SBE detection. By adding only one universal primer, equal PCR efficiency for all amplicons in a high quantity PCR step can be ensured. (iii) Detection. Specific APEX-2 oligonucleotides (also used in the first phase) are immobilized by modifying the previously activated array surface. After specific hybridization, the immobilized primer is extended by one labeled dideoxynucleotide (terminator), which is complementary to the nucleotide of the allele studied. APEX-2, primer design and slide preparation were slightly modified from Krjutskov et al. [24]. In the first phase, 5 ml genomic

3 K. Krjutškov et al. / Forensic Science International: Genetics 4 (2009) Fig. 1. The APEX-2 principle and the lengths of binding regions. (A) APEX-2 primers (APEX-2 primers 1 and 2) bind to genomic DNA sequences immediately upstream of the position of interest (SNP/mutation). After primer extension, the synthesized sequence contains the complementary strand of the respective APEX-2 primer and the position of interest. (B) The universal primer hybridizes to the 3 0 end of the previously generated product during template amplification. (C) The purified universal phase PCR product hybridizes to the immobilized APEX-2 primers that have a 5 0 -amino modification, enabling them to be spotted and immobilized on the array surface. Genotyping is performed as a four-color single-base extension reaction. (D) Histogram represents distribution of lengths of 124 specific regions for primer binding. DNA ( ng/ml) was dehydrated by heating at 95 8C for 5 min. Multiplex PCR was carried out in a 5-mL volume containing PCR buffer: 60 mm Tris HCl ph 8.3, 60 mm KCl, 15 mm (NH 4 ) 2 SO 4 ), 0.35 mm each dntp (N = G, C, A, T) (Fermentas), 4.75 mm MgCl 2, 1 U TrueStart TM Taq DNA polymerase (Fermentas), 2% DMSO (Sigma Aldrich Chemie, Taufkirchen, Germany), 450 nm each Y chromosome specific primer, 30 nm each mitochondrial primer and 95 nm each autosomal primer (Metabion International AG, Martinsried, Germany). Five microliters of multiplex PCR mixture was directly added to a previously denatured and dewatered DNA probe, mixed for 3 5 s and centrifuged. Five microliters of mineral oil (Sigma Aldrich Chemie) was added before the final spin. Thirty cycles of PCR were performed in a TProfessional Basic (Biometra, Göttingen, Germany) under the following conditions: 98 8C for 40 s (initial denaturation); 30 cycles of 95 8C/20 s (denaturation), 61 8C/ 20 s, 62 8C/20 s, 63 8C/20 s, 64 8C/20 s, 65 8C/40 s, 67 8C/40 s (sixstep annealing) and 72 8C/20 s (extension) without lid heating. Universal primer amplification, product purification, SBE on the oligoarray and signal detection were carried out as described previously [24]. Five genotype controls were performed for the APEX-2 forensic assay, as previously described in the APEX-1 protocol [25] Detection of genotyping errors and Hardy Weinberg disequilibrium Seventy-nine DNA samples were genotyped and allele frequencies were estimated by the allele counting method. The goodness-of-fit x 2 -test was used to identify departure from Hardy Weinberg equilibrium for each polymorphism. The presence of Mendelian inconsistencies were estimated using data from 17 analyzed family trios, where genotypes from mother, father and child(ren) were compared to find potential genotyping or inheritance errors. Haploview Ver. 4.1 software was used for all analyses. 3. Results and discussion 3.1. Assay validation and allele frequencies The success of genotyping by the initial 132-plex APEX-2 forensic assay is summarized in Table 1A. After the preliminary experiments and optimization procedure, eight markers (Supplementary Table 1) were removed from analysis owing to low call rate (50 90%) and/or high background signal. As a result, the assay s success rate was 94%. The optimized 124-plex assay was tested with 101 DNAs including 17 families (56 DNAs), five internal controls with previously known genotypes and 79 unrelated individuals. The rate of genotyping errors was assessed in two ways: first by comparing family genotypes by Y chromosome patrilineal, mitochondrial matrilineal and autosomal analysis, following Mendelian rules; second, on the basis of five individuals previously typed by the APEX-1 assay. Family scanning of 1783 Table 1A Classification of analyzed DNA samples. Number of analyzed DNAs 101 Unrelated DNAs 79 Related (children) 22 Families 17 Males 63 Females 38

4 46 Table 1B Quality parameters for 124-plex assay. K. Krjutškov et al. / Forensic Science International: Genetics 4 (2009) Table 2 Results from the analysis of the mitochondrial and Y chromosome haplogroups. Value Counts Y chromosome Call rate and missing calls Y-chromosome 99.89% 2 of 1,827 Mitochondrion 99.62% 19 of 4,949 Autosomes 99.61% 18 of 4,646 Total 99.66% 39 of 11,422 Concordance rate with 99.82% 1 of 562 other method Mendelian consistency 99.83% 3 of 1,783 positions exposed three Mendelian inconsistencies (99.83%): autosomal rs907100, rs and rs where child heterozygosity was not detected automatically (Table 1B). For the internal control DNAs, one typing error was detected within 562 comparisons (99.82%). Hence, the autosomal SNP concordance rate, across family trios and internal controls, was estimated as 99.83% (4 errors out of 2345 comparisons (1783 (family data) (internal controls)). The estimated call rates in the 101 individuals were 99.89%, 99.62% and 99.61%, respectively, for the Y chromosome, mitochondrial and autosomal markers. In summary, 39 out of 11,422 genotypes were not typed (overall call rate 99.66%). Nearly half the no-call positions (18 out of 39) occurred in four markers where, in addition to amplification failure, oligoarray spotting reproducibility might have caused numerous missing calls. The allele frequencies of the Y chromosome and mitochondrial markers are shown in Supplementary Table 1. Sixteen of the 29 Y chromosome markers (55%) represented only one allele, where minor allele frequency (MAF) was 0. This result is not surprising given that the invariable SNPs define haplogroups that are uncommon in northern Europe where our samples were collected. Consistent with our regional sampling, the highest MAF values were measured for markers rs (0.438, haplogroup P), rs3900 (0.36, haplogroup K) and rs (0.36, haplogroup IJ). In 36 of the 49 mitochondrial positions (73%), two alleles were detected with the highest MAF value of in position Transition at this position defines haplogroup H which is the most frequent haplogroup throughout Europe. Allele frequencies and Hardy Weinberg equilibrium P-values for autosomal markers are shown in Supplementary Table 1. Slight deviations from HW expectations were found for SNPs rs and rs (P = 0.02), possibly because of random sampling error caused by the relatively small number (79) of unrelated genotyped individuals Mitochondrial DNA and Y chromosome haplogroup frequencies The assay characterized 49 informative SNP markers of mtdna selected to identify major European (H K, T X), African (L0 L3) and Asian (M) haplogroups, the phylogenetic relationships of which are depicted in Supplementary Fig. 1A. The genotyping results of the 79 unrelated North European individuals, 64 Estonians and 15 Swedes, are summarized in Table 2; 24 different haplogroups are identified, with the three most frequent, H1 (17.7%), H* (13.9%) and J1c (10.1%), accounting for more than 40% of the total variation. In general, the haplogroup profile obtained from the composite sample of individuals of Swedish and Estonian origin appeared to be consistent with the expected haplogroup frequency in Northeast Europe [26,27]. Only three individuals displayed haplogroup affiliations atypical of Europe two of them sharing haplogroup M specific markers, which is the most common lineage group in Asia, and one left unclassified at the level of haplogroup L3, potentially indicating African maternal origin. Haplogroup n % of sample Origin (Est/Swe) I % 9/6 I2b % 0/3 K* 1 2.0% 0/1 N % 10/0 P&Q* 1 2.0% 0/1 R1a % 14/1 R1b % 4/1 Total % 37/13 Mitochondrial DNA Haplogroup n % of sample L % M 2 2.5% W 3 3.8% X 1 1.3% N1a 1 1.3% I 1 1.3% R* 1 1.3% H* % H % H1b 4 5.1% H2a 1 1.3% H % J1a 1 1.3% J1c % J2a % T % T2b 5 6.3% U % U % U % U5a 5 6.3% U5b 3 3.8% U8* 1 1.3% K % Total % *Intermediate-level haplogroup determined. Similarly, the Y chromosome haplogroup profile, with haplogroups I1, N3, R1a and R1b together describing 90% of the variation, is consistent with the Northeast European origin of the samples [27 29]. The 29 Y chromosome markers that we used were unable to yield specific haplogroup classification for only two individuals, who were defined up to the level of major haplogroups K* and P*, which are widespread across continents (Table 2). For 73 of the 79 individuals, the haplogroup affiliations were identified unambiguously, while six DNAs required additional analysis to control/confirm the assay Required amount of DNA To measure the required concentration of starting material and to determine the approximate concentration threshold, five total DNAs (isolated from males by the high salt precipitation method) were analyzed in the series 100; 50; 25; 12.5, 1 and 0.5 ng per reaction. The call rates detected over the assay were 99.68%, 99.19%, 98.39%, 95%, 83% and 79%, respectively (Fig. 2). For comparison, mitochondrial positions gave high outcomes over all concentrations (>95% call rate). Thirty percent (23 out of 75) of Y chromosome and autosomal SNPs generated false calls in concentrations lower than 25 ng per reaction. However, the corresponding figure for mitochondrial DNA was 8% (4 out of 49) at a concentration of 0.5 ng per reaction (Fig. 2). Therefore, we can conclude that in order to achieve a call rate over 99%, the

5 K. Krjutškov et al. / Forensic Science International: Genetics 4 (2009) Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi: /j.fsigen References Fig. 2. Graphical summary shows the relationship between call rate values, false signal appearance and various DNA concentrations. Two categories, mitochondrial and genomic positions (autosomal and Y chromosome), are depicted. amount of isolated total DNA for mitochondrial positions can be roughly 50 times lower than for autosomal positions, where 50 ng of total DNA is needed. In comparison to the 52 SNP-multiplex PCR assay [4] where ng of DNA is required for autosomal SNP analysis, a greater amount of DNA is needed for the APEX-2 assay. The APEX-2 assay requires the simultaneous amplification of autosomal, Y chromosome and mitochondrial targets that are not present in equal starting concentrations. The wide range of target copies can provide a significant challenge for multiplex PCR assays. 4. Conclusion The results of the optimization and validation experiments for the APEX-2 forensic assay have shown that the protocol has high reliability and is able to amplify and analyze 124 genetic markers from three different targets (mitochondria, Y chromosome and autosomes) simultaneously in one reaction. While composing a marker set that worked well, we removed eight SNPs in the early optimization step. Six of these belonged to the autosomal, one to the mitochondrial and one to the Y chromosome marker set. In forensic analysis it is generally necessary to engage new efficient and highly polymorphic markers for autosomal assortment, but this was not a goal in the present study. The assay was very successful in mitochondrial positions, and throughout the experiments, mitochondrial array signals were clearly more intense than autosomal or Y chromosomal calls (data not shown). In summary, further extension of the application of APEX-2 to the Y chromosomal and mitochondrial sectors provided knowledge about the experimental conditions. As a result of this paper, a new and promising microarray-based assay has been developed for forensic applications. The throughput and sensitivity of the 124-plex APEX-2 enables forensic samples to be analyzed for genealogical and justice databases if a saliva or blood sample is available and 50 ng of starting material is attainable. Acknowledgments We would like to thank Triinu Temberg for technical assistance and Jüri Parik for helpful suggestions in study design. Microarrays used in this study were kindly printed for us by Asper Biotech (Tartu, Estonia) that also provided funds for ordering oligonucleotides. This work was supported by the project of GARLA 6808, Targeted Financing from the Estonian Government (SF As08) and by the European Union through the European Regional Development Fund. [1] C. Børsting, J.J. Sanchez, N. 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