Forensic Science International: Genetics 5 (2011) e89–e92
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Forensic Science International: Genetics journal homepage: www.elsevier.com/locate/fsig
Forensic Population Genetics—Letter to the Editor Swedish population data and concordance for the kits PowerPlex1 ESX 16 System, PowerPlex1 ESI 16 System, AmpFlSTR1 NGMTM, AmpFlSTR1 SGM PlusTM and Investigator ESSplex
1. Population Samples were obtained from 425 unrelated individuals with Swedish surnames after informed consent and made anonymous before they were analysed for a Swedish database. Samples were collected from four different locations in Sweden; Lund, Linko¨ping, Uppsala and Umea˚. 2. Extraction and quantification Genomic DNA was isolated from blood samples using a phenol/ chloroform extraction procedure according to Smith et al. [1] with the modification that Nonidet P-40 was used instead of SDS. The extracted DNA was quantified using ABI 7300 Real Time PCR System and the chemistry of QuantifilerTM Human DNA Quantification Kit (Applied Biosystems, USA) following manufacturer’s instructions. 3. PCR amplification Samples were amplified using 0.25–1.25 ng target DNA depending on the manufacturer’s recommendations. The amplification of the autosomal STRs was performed using reaction conditions as described by each manufacturer. The samples were amplified in a GeneAmp PCR System 9700 (Applied Biosystems, USA). 4. Electrophoresis and typing The amplified products were detected with the ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems, USA) using the filter set recommended by each manufacturer. Allele calling was performed using GeneMapperID v 3.2 (Applied Biosystems, USA). The allelic ladder included in each kit was used as a reference. GeneScan1 400HD [ROX] was used as size standard for AmpFlSTR1 SGM PlusTM (Applied Biosystems, USA), CC5 Internal Lane Standard 500 for PowerPlex1 ESX 16 System and PowerPlex1 ESI 16 System (Promega Corporation, USA), GeneScanTM -500 LIZ1 for AmpFlSTR1 NGMTM (Applied Biosystems, USA) and DNA size standard 550 (BTO) for Investigator ESSplex (Qiagen, Germany). 5. Quality control The biology unit at SKL is accredited according to ISO/IEC 17025 and participates in FID (College of American Pathologists Identity 1872-4973/$ – see front matter ß 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigen.2010.11.005
Programme), CTS (Collaborative Testing Services Inc.) and GEDNAP proficiency tests. 6. Analysis of data Calculations of allele frequencies, power of discrimination and power of exclusion were carried out using PowerStats version 12 [2]. Observed and expected heterozygosity and P-values of the Hardy–Weinberg equilibrium tests were assessed using GDA version 1.0 [3]. Concordance evaluation has been performed using Microsoft Excel and R [4]. 7. Results One of the obtained profiles in the study was neglected due to the presence of three alleles in the vWA loci for all five kits tested. All statistical evaluations and calculations were therefore made on the remaining 424 genotype profiles based on the data obtained for the PowerPlex1 ESX 16 System. Allele frequencies, values for observed and expected heterozygosity, P-values for Hardy– Weinberg equilibrium, power of discrimination and power of exclusion for PowerPlex1 ESX 16 System loci, in this Swedish population of 424 individuals, are presented in Table 1. The concordance study [5] made for this population data only revealed four discordant alleles between the kits (Table 2). 8. Other remarks No evidence of deviations from Hardy–Weinberg equilibrium were found, when using a Bonferroni correction (P < 0.05/ 15 = 0.0033) or the usual threshold of statistical significance at 0.05. The observed heterozygosity varies between 0.755 for TH01 and 0.892 for D1S1656. The power of discrimination was smallest for D22S1045; 0.869, and largest for D1S1656; 0.982. The power of exclusion ranges from 0.518 for TH01 to 0.778 for D1S1656. One male sample revealed a null allele (X) in Amelogenin when amplifying with AmpFlSTR1 NGMTM but not when using AmpFlSTR1 SGM PlusTM (Fig. 1, Table 2). According to the manufacturer both kits are composed of identical primer sequences for the same loci. However, electrophoretic mobility changes and increased amplification success can be achieved without altering the primer sequences, e.g. by using mobility modifiers [6,7] and locked nucleic acids (LNAs) [8,9]. LNAs increase the specificity of primer binding, and LNA containing primers may therefore be more sensitive to mutations in the primer binding site. If such primer modifications are used in the Amelogenin marker of the AmpFlSTR1 NGMTM kit it could be an explanation for the missing X allele in one of the samples. Possibly the dropout is extorted by a mutation in, or adjacent to, the primer site sequence, not affecting amplification with the non-modified primers used in AmpFlSTR1 SGM PlusTM.
Letter to the Editor / Forensic Science International: Genetics 5 (2011) e89–e92
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Table 1 Allele frequencies for PowerPlex1 ESX 16 System loci in a Swedish population based on 424 individuals. Allele
D3S1358
TH01
D21S11
D18S51
D10S1248
D1S1656
D2S1338
D16S539
D22S1045
vWA
D8S1179
FGA
D2S441
D12S391
D19S443
5 6 7 8 9 9.3 10 11 11.3 12 12.2 12.3 13 13.2 14 14.2 14.3 15 15.2 15.3 16 16.2 16.3 17 17.1 17.2 17.3 18 18.3 19 19.3 20 21 22 22.2 23 23.2 24 24.2 25 25.2 26 27 28 28.1 29 29.2 29.3 30 30.2 31 31.2 32 32.2 33 33.2
– – – – – – – 0.006 – – – – 0.004 – 0.106 – – 0.274 – – 0.241 – – 0.229 – – – 0.131 – 0.009 – 0.001 – – – – – – – – – – – – – – – – – – – – – – – –
0.001 0.195 0.191 0.092 0.157 0.357 0.007 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – 0.001 0.001 0.001 0.001 0.034 0.186 0.001 0.184 0.001 0.002 0.257 0.051 0.081 0.073 0.011 0.081 0.001 0.031
– – – – – – 0.011 0.004 – 0.138 – – 0.121 – 0.199 – – 0.138 – – 0.117 – – 0.106 – – – 0.072 – 0.047 – 0.022 0.015 0.006 – 0.002 – 0.001 – – – – – – – – – – – – – – – – – –
– – – – – – – 0.007 – 0.027 – – 0.301 – 0.310 – – 0.206 – – 0.120 – – 0.027 – – – 0.001 – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – 0.007 0.077 – 0.132 – – 0.052 – 0.080 – 0.001 0.103 – 0.081 0.104 – 0.059 0.048 0.001 – 0.147 0.009 0.085 – 0.013 – – – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – – – – – – – – – – – – – – 0.031 – – 0.198 – – – 0.099 – 0.112 – 0.156 0.021 0.037 – 0.093 – 0.113 – 0.113 – 0.022 0.005 – – – – – – – – – – – – –
– – – 0.009 0.120 – 0.041 0.300 – 0.316 – – 0.176 – 0.035 – – 0.002 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – 0.134 – 0.012 – – 0.005 – 0.047 – – 0.354 – – 0.356 – – 0.085 – – – 0.007 – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – – – – – – 0.001 – 0.094 – – 0.090 – – 0.210 – – 0.265 – – – 0.242 – 0.079 – 0.017 0.002 – – – – – – – – – – – – – – – – – – – – – – –
– – – 0.011 0.008 – 0.088 0.083 – 0.131 – – 0.358 – 0.226 – – 0.070 – – 0.022 – – 0.002 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – – – – – – – – – – – – – – – – – 0.001 – – – 0.026 – 0.053 – 0.175 0.180 0.170 0.015 0.132 0.004 0.142 – 0.074 – 0.025 0.004 – – – – – – – – – – – – –
– – – 0.004 – – 0.188 0.375 0.051 0.038 – 0.001 0.028 – 0.270 – – 0.041 – – 0.005 – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – – –
– – – – – – – – – – – – – – – – – 0.052 – – 0.019 – – 0.110 – – 0.025 0.189 0.008 0.110 0.002 0.114 0.117 0.117 – 0.087 – 0.038 – 0.008 – 0.004 0.001 – – – – – – – – – – – – –
– – – – – – – 0.002 – 0.072 0.001 – 0.200 0.024 0.374 0.019 – 0.188 0.032 – 0.058 0.022 – 0.005 – 0.002 – 0.001 – – – – – – – – – – – – – – – – – – – – – – – – – – – –
Obs. Ha Exp. Hb Pc PDd PEe
0.816 0.787 0.204 0.913 0.629
0.755 0.766 0.387 0.908 0.518
0.818 0.843 0.774 0.957 0.634
0.858 0.875 0.241 0.969 0.712
0.795 0.756 0.793 0.892 0.589
0.892 0.906 0.457 0.982 0.778
0.873 0.878 0.953 0.971 0.740
0.757 0.763 0.946 0.909 0.522
0.759 0.721 0.822 0.869 0.526
0.802 0.805 0.972 0.935 0.603
0.792 0.784 0.497 0.921 0.585
0.833 0.862 0.152 0.965 0.661
0.776 0.746 0.337 0.894 0.555
0.866 0.888 0.347 0.975 0.726
0.816 0.775 0.292 0.918 0.629
a b c d e
Observed heterozygosity. Expected heterozygosity. P-value Hardy–Weinberg equilibrium exact test based on 10,000 shufflings. Power of discrimination. Power of exclusion.
In one sample a null allele (13) in D18S51 was revealed when amplifying with AmpFlSTR1 NGMTM and AmpFlSTR1 SGM PlusTM (Table 2). The two kits contain identical primer sequences and the null allele is consistent with a mutation in a primer binding site [10].
When amplified with Investigator ESSplex, one sample revealed a microvariant (11.3) in D8S1179, whereas the other kits presented allele 12 (Table 2). This discrepancy may be due to a deletion in the flanking region, outside of the ESSplex primer sites and exterior to the regions being amplified by the other kits [10,11].
Letter to the Editor / Forensic Science International: Genetics 5 (2011) e89–e92
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Table 2 Discordant typing results between the five kits analysed. Null alleles are indicated with a minus sign. Different alleles are shown in bold. These samples were re-typed to verify the results. Locus
AMPFlSTR1 SGM PlusTM
AmpFlSTR1 NGMTM
PowerPlex1 ESX 16 System
PowerPlex1 ESI 16 System
Investigator ESSplex
AMELOGENIN D18S51 D8S1179
X/Y 12/ 8/12
/Y 12/ 8/12
X/Y 12/13 8/12
X/Y 12/13 8/12
X/Y 12/13 8/11.3
[()TD$FIG]
Fig. 1. The amelogenin marker for the male sample with a missing X allele in AmpFlSTR1 NGMTM (a), but present using AmpFlSTR1 SGM PlusTM (b).
This paper follows the guidelines for publication of population data requested by the journal [12]. References [1] J.C. Smith, C.R. Newton, A. Alves, R. Anwar, D. Jenner, Highly polymorphic minisatellite DNA probes. Further evaluation for individual identification and paternity testing, J. Forensic Sci. Soc. 30 (1990) 3–18. [2] PowerStats Version 12, Promega Corporation Website. Available from: http:// www.promega.com/geneticidtools/powerstats/. [3] P.O. Lewis, D. Zaykin, Genetic Data Analysis: computer program for the analysis of allelic data, Version 1.0 (d16c), 2001. Available from: http://lewis.eeb.uconn.edu/ lewishome/software.html. [4] The R Foundation for Statistical Computing. Available from: http://www.rproject.org. [5] C.R. Hill, D.L. Duewer, J.M. Butler, Strategies for Concordance Testing, Profiles in DNA 13(1) [Internet], 2010. Available from: www.promega.com/profiles/1301/ 1301_08.html (cited: 2010-08-13).
[6] P.D. Grossman, W. Bloch, E. Brinson, C.C. Chang, F.A. Eggerding, S. Fung, D.A. Iovannisci, S. Woo, E.S. Winn-Deen, High-density multiplex detection of nucleic acid sequences: oligonucleotide ligation assay and sequence-coded separation, Nucleic Acids Res. 22 (1994) 4527–4534. [7] V. Barbier, J.-L. Viovy, Advanced polymers for DNA separation, Curr. Opin. Biotechnol. 14 (1) (2003) 51–57. [8] K.N. Ballantyne, R.A.H. van Oorschot, R.J. Mitchell, Increased amplification success from forensic samples with locked nucleic acids, Forensic Sci. Int. Genet. (2010), doi:10.1016/j.fsigen.2010.04.001. [9] J.J. Mulero, L.K. Hennessy, Method and compositions for nucleic acid amplification, U.S. Patent 0004662 (2009). [10] C.R. Hill, D.L. Duewer, M.C. Kline, C.J. Sprecher, R.S. McLaren, D.R. Rabbach, B.E. Krenke, M.G. Ensenberger, P.M. Fulmer, D.R. Storts, J.M. Butler, Concordance and population studies along with stutter and peak height ratio analysis for the PowerPlex1 ESX 17 and ESI 17 Systems, Forensic Sci. Int. Genet. (2010), doi:10.1016/j.fsigen.2010.03.014. [11] C.R. Hill, M.C. Kline, J.J. Mulero, R.E. Lagace´, C.-W. Chang, L.K. Hennessy, J.M. Butler, Concordance study between the AmpFlSTR1 MiniFilerTM PCR amplification kit and conventional STR typing kits, J. Forensic Sci. 52 (2007) 870–873.
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Letter to the Editor / Forensic Science International: Genetics 5 (2011) e89–e92
[12] A´. Carracedo, J.M. Butler, L. Gusma˜o, W. Parson, L. Roewer, P.M. Schneider, Publication of population data for forensic purposes, Forensic Sci. Int. Genet. 4 (2010) 145–147.
Linda Albinsson Lina Nore´n Ronny Hedell Swedish National Laboratory of Forensic Science (SKL), SE-581 94 Linko¨ping, Sweden Ricky Ansell a, b, * Swedish National Laboratory of Forensic Science (SKL), SE-581 94 Linko¨ping, Sweden
a
b
Department of Physics, Chemistry and Biology (IFM), Linko¨pings Universitet, Linko¨ping, Sweden
*Corresponding
author at: Swedish National Laboratory of Forensic Science (SKL), Brigadgatan, SE-581 94 Linko¨ping, Sweden. Tel.: +46 10 562 81 19; fax: +46 10 562 80 43 E-mail address:
[email protected] (R. Ansell) 2 September 2010