- Email: [email protected]
Forensic and population genetic analysis of Serbian population using 21 STR loci of GlobalFiler™ PCR amplification kit
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/fsigss
Forensic and population genetic analysis of Serbian population using 21 STR loci of GlobalFiler™ PCR amplification kit ⁎
Dragana Zgonjanina,b, , Reem Almheiric, Rashed Alghafric,d, Dalibor Nediće,f, Goran Stojiljkovića,b a
Institute of Forensic Medicine, Clinical Center of Vojvodina, Novi Sad, Serbia Faculty of Medicine, University of Novi Sad, Novi Sad, Serbia c General Department of Forensic Sciences and Criminology, Dubai Police G.H.Q., Dubai, United Arab Emirates d College of Science, Biology Department, United Arab Emirates University, United Arab Emirates e Institute of Forensic Medicine of Republic of Srpska, Banja Luka, Bosnia and Herzegovina f Medical Faculty, University of Banja Luka, Banja Luka, Republic of Srpska, Bosnia and Herzegovina b
A R T I C LE I N FO
A B S T R A C T
Keywords: Autosomal STRs Serbia population Globalfiler Forensics
Autosomal short tandem repeats (STRs) have been widely used in forensic investigations. Prior to the application of any DNA based identification method, it is essential to estimate the allele frequencies and forensic statistical parameters of targeted STR loci in each population in order to provide a more precise reference database for forensic investigation. The GlobalFiler™ Kit is a multiplex assay that combines the 13 original CODIS loci with 7 non-overlapping loci from the expanded European Standard Set (ESS), as well as the highly discriminating SE33 locus, two Y-based loci and the sex determining maker, Amelogenin. The full complement of loci in the GlobalFiler™ Kit are: D13S317, D7S820, D5S818, CSF1PO, D1S1656, D12S391, D2S441, D10S1248, D18S51, FGA, D21S11, D8S1179, vWA, D16S539, TH01, D3S1358, AMEL, D2S1338, D19S433, DYS391, TPOX, D22S1045, SE33 and a Y-specific insertion/deletion locus (Yindel). The 6-dye GlobalFiler™ PCR Amplification kit (ThermoFisher Scientific) comprises 21 autosomal STRs have already been proven to be able to provide reliable DNA profiling results and enhance the power of discrimination between individuals. In this study, we are presenting an analysis of GlobalFiler STR loci on 209 unrelated individuals from Serbia.
1. Introduction Evaluating STR allelic frequencies is an essential prerequisite to start applying such analysis in the forensic casework, as it has been shown before that, each population will have its own composition of alleles distribution and therefore, it will facilitate the correct calculations of weigth of evidence. By utilizing one of the most powerful commercially available STR amplification kits (GloblaFiler™) can aid development of allelic frequency database to enhance knowledge about patterns of genetic diversity and to have further understanding about population genetic structure [1].
DNA was amplified using the GlobalFiler™ Kit (ThermoFisher Scientific) in a thermal cycler GeneAmp 9700 PCR system (Applied Biosystems), according to manufacturer’s recommendations, but reducing the PCR final volume to half (12.5 μl) of the recommended quantity. The electrophoresis was carried out on 3500 Genetic Analyzer (Applied Biosystems, USA) and the fragment analysis was performed with GeneMapper ID-X v.1.4 software (Applied Biosystems). Arlequin version 3.5 [3] was used to calculate allele frequencies, expected heterozygosity (He), observed heterozygosity (Ho), probability value of the HardyWeinberg equilibrium (HWE) exact test. Population genetics statistical parameters were calculated using PowerStats version 1.2.
2. Materials and methods
3. Results
Total of 209 DNA samples was extracted and purified from blood stains or buccal swabs by using the Chelex 100 extraction method [2] from unrelated individuals from Serbia, following informed consent.
Allele frequencies and resulting statistical parameters are given in Table 1. No significant deviations from Hardy–Weinberg equilibrium and linkage disequilibrium were detected within and between the 21
⁎
Corresponding author at: Institute of Forensic Medicine, Clinical Center of Vojvodina, Novi Sad, Serbia. E-mail addresses: [email protected], [email protected] (D. Zgonjanin).
https://doi.org/10.1016/j.fsigss.2019.09.020 Received 10 September 2019; Accepted 21 September 2019 1875-1768/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Dragana Zgonjanin, et al., Forensic Science International: Genetics Supplement Series, https://doi.org/10.1016/j.fsigss.2019.09.020
6 6.2 7 8 9 9.3 10 11 12 13 13.2 14 14.2 14.3 15 15.2 15.3 16 16.2 16.3 17 17.2 17.3 18 18.2 18.3 19 19.2 19.3 20 20.2 20.3 21 21.2 22 22.2 23 23.2 24 24.2 25 25.2 26 26.2 27 27.2 28 28.2 29 29.2 30 30.2 31
Allele/ Locus
0.222
0.278
0.187
0.242
0.268
0.206
0.167
0.019
0.100
0.093
2 0.177 0.005 0.184 0.010 0.144 0.002 0.120
0.105 0.002
0.108 0.002
0.022
FGA
0.208 0.041 0.055
0.196
0.036
0.002
0.251 0.153 0.093 0.196 0.285 0.019 0.002
TH01
0.167
0.002
0.002 0.005
0.043 0.031
0.165 0.038
0.014 0.096 0.232 0.012 0.321 0.038
D19S433
0.005
0.007
0.045
0.268
0.335 0.067 0.053 0.026
0.002 0.196
D2S441
0.053 0.005 0.050 0.002 0.007
0.002
0.010
0.002
0.074
0.112
0.139
0.139 0.002
0.191
0.007 0.005 0.134 0.136
D18S51
0.014
D21S11
0.017
0.017
0.139
0.225
0.055 0.074 0.144 0.323
0.012 0.012
D8S1179
0.033
0.057 0.280 0.017
0.010 0.555 0.079
0.002
TPOX
0.081
0.007
0.033
0.112
0.005
0.007
0.285 0.297 0.325 0.053
CSF1PO
0.067 0.292 0.301 0.148
D16S539
0.002 0.031
vWA
0.014 0.136
D3S1358
Table 1 Allele frequencies and statistical parameters of 21 autosomal STRs loci in a population sample from Serbia (N = 209).
0.148
0.002
0.024
0.100
0.285
0.388
0.148 0.022 0.031
D22S1045
0.007
0.007
0.069 0.333 0.380 0.160
0.043
D5S818
0.026
0.062 0.361 0.268 0.089
0.139 0.055
D13S317
0.007
0.282 0.177 0.129 0.024
0.026 0.175 0.179
D7S820
0.046 0.002
0.046
0.087
0.058
0.043
0.048
0.029
0.048
0.024 0.002 0.007 0.041
0.072 0.010
0.060 0.002
0.077 0.002
0.077
0.063
0.055
0.024 0.002
0.005 0.010
SE33
0.002
0.010
0.029
0.175
0.234
0.266
0.007 0.022 0.256
D10S1248
0.026
0.100 0.002 0.199
0.041 0.141 0.053 0.055 0.100 0.002
0.007
0.012
0.100
0.105
(continued on next page)
0.002
0.017
0.012
0.110
0.065
0.002 0.093
0.017
0.029
0.014 0.134
0.014
0.105
0.146
0.033 0.129
0.060
0.086
0.093
0.234
0.053
0.002
0.065
0.002 0.134
D2S1338
0.005
D12S391
0.072
0.112 0.132 0.081
D1S1656
D. Zgonjanin, et al.
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
0.965 0.785 4.019 0.876
D12S391
0.961 0.785 4.750 0.895
D1S1656
0.019 0.010 0.002 0.005 0.002 0.990 0.785 4.750 0.895
0.019
0.915 0.488 1.900 0.737
D10S1248
0.898 0.472 1.833 0.727 0.862 0.411 1.608 0.689
0.932 0.588 2.430 0.794
4. Discussion In sum, among the 21 markers presents in the commercial kit GlobalFiler™ the most polymorphic, informative and powerful locus in forensic identification was found to be SE33. This marker has the highest PIC, PD, PE, PI and the lowest MP and homozygosity frequency. These results are concordant to others studies, that showed that TPOX is the molecular marker, present in CODIS, that revealed lowest variation among individuals [4].
0.964 0.652 2.903 0.828 0.843 0.434 1.685 0.703
0.887 0.496 1.935 0.742
5. Conclusion
0.898 0.537 2.133 0.766 0.876 0.441 1.713 0.708 0.965 0.717 3.603 0.861 0.941 0.615 2.613 0.809 0.922 0.615 2.613 0.809
0.936 0.571 2.322 0.785
0.914 0.580 2.375 0.790
0.875 0.426 1.659 0.699
0.791 0.245 1.136 0.699
0.931 0.597 2.488 0.799
0.005
0.120 0.007 0.117 0.043
31.2 32 32.2 33.2 34 34.2 35 PD PE PI Ho
This study represents the first report of frequencies for the GlobalFiler™ markers set in a population of Serbia. Power of discrimination, gene diversity and alleles frequencies presented in this study suggest that the 21 STR loci have great value in forensic casework analysis as well as in kinship analysis. PD – power of discrimination, PE – power of exclusion, PI –paternity index, Ho – observed heterozygosity.
SE33 D7S820 D13S317 D5S818 D22S1045 FGA TH01 D19S433 D2S441 D18S51 D21S11 D8S1179 TPOX CSF1PO D16S539 vWA D3S1358 Allele/ Locus
Table 1 (continued)
STR loci. Locus SE33 has shown the greatest power of discrimination in Serbia population, whereas TPOX has was the lowest. The combined power of discrimination was found to be 0.99999999999999999999999989954, whereas the random match probability was found to be 4.2 × 10−26 and the combined power of exclusion was 0.999999990605. Relative to the power of discrimination (PD), the values range between 0.7911 and 0.9899, being the highest and the lowest values given, respectively, by SE33 and TPOX markers. The most informative marker is SE33, which present the highest polymorphic information content (PIC) of 0.941. All of the other markers have PIC values above 0.547. Moreover, the power of exclusion (PE) is 0.2454 for TPOX marker (minimum value) and 0.7847 for SE33 (maximum value). Paternity Index (PI) is situated between 1.1358 (TPOX) and 4.75 (SE33).
0.965 0.597 2.824 0.823
D2S1338
D. Zgonjanin, et al.
Declaration of Competing Interest No conflicts of interest. Acknowledgements The project was supported by Vojvodine Government, by the Grant No. 142-451-2704/2018-01/02 of the Provincial Secretariat for higher education and scientific research of the Vojvodina Province. References [1] J. Buckleton, J. Curran, J. Goudet, D. Taylor, A. Thiery, B.S. Weir, Population-specific FST values for forensic STR markers: a worldwide survey, Forensic Sci. Int. Genet. 23 (2016) 91–100, https://doi.org/10.1016/j.fsigen.2016.03.004. [2] P.S. Walsh, D.A. Metzger, R. Higuchi, Chelex 100 as a medium for simple extraction of DNA for PCR-based typing for forensic material, Biotechniques 10 (1991) 506–513. [3] L. Excoffier, H.E. Lischer, Arlequin suite ver 3. 5: a new series of programs to perform population genetics analyses under Linux and Windows, Mol. Ecol. Resour. 10 (2010) 564–567. [4] P. Thanakiatkrai, T. Kitpipit, Current STR-based techniques in forensic science, Maejo Int. J. Sci. Technol. 7 (2013) 1–15.
3
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/fsigss
Forensic and population genetic analysis of Serbian population using 21 STR loci of GlobalFiler™ PCR amplification kit ⁎
Dragana Zgonjanina,b, , Reem Almheiric, Rashed Alghafric,d, Dalibor Nediće,f, Goran Stojiljkovića,b a
Institute of Forensic Medicine, Clinical Center of Vojvodina, Novi Sad, Serbia Faculty of Medicine, University of Novi Sad, Novi Sad, Serbia c General Department of Forensic Sciences and Criminology, Dubai Police G.H.Q., Dubai, United Arab Emirates d College of Science, Biology Department, United Arab Emirates University, United Arab Emirates e Institute of Forensic Medicine of Republic of Srpska, Banja Luka, Bosnia and Herzegovina f Medical Faculty, University of Banja Luka, Banja Luka, Republic of Srpska, Bosnia and Herzegovina b
A R T I C LE I N FO
A B S T R A C T
Keywords: Autosomal STRs Serbia population Globalfiler Forensics
Autosomal short tandem repeats (STRs) have been widely used in forensic investigations. Prior to the application of any DNA based identification method, it is essential to estimate the allele frequencies and forensic statistical parameters of targeted STR loci in each population in order to provide a more precise reference database for forensic investigation. The GlobalFiler™ Kit is a multiplex assay that combines the 13 original CODIS loci with 7 non-overlapping loci from the expanded European Standard Set (ESS), as well as the highly discriminating SE33 locus, two Y-based loci and the sex determining maker, Amelogenin. The full complement of loci in the GlobalFiler™ Kit are: D13S317, D7S820, D5S818, CSF1PO, D1S1656, D12S391, D2S441, D10S1248, D18S51, FGA, D21S11, D8S1179, vWA, D16S539, TH01, D3S1358, AMEL, D2S1338, D19S433, DYS391, TPOX, D22S1045, SE33 and a Y-specific insertion/deletion locus (Yindel). The 6-dye GlobalFiler™ PCR Amplification kit (ThermoFisher Scientific) comprises 21 autosomal STRs have already been proven to be able to provide reliable DNA profiling results and enhance the power of discrimination between individuals. In this study, we are presenting an analysis of GlobalFiler STR loci on 209 unrelated individuals from Serbia.
1. Introduction Evaluating STR allelic frequencies is an essential prerequisite to start applying such analysis in the forensic casework, as it has been shown before that, each population will have its own composition of alleles distribution and therefore, it will facilitate the correct calculations of weigth of evidence. By utilizing one of the most powerful commercially available STR amplification kits (GloblaFiler™) can aid development of allelic frequency database to enhance knowledge about patterns of genetic diversity and to have further understanding about population genetic structure [1].
DNA was amplified using the GlobalFiler™ Kit (ThermoFisher Scientific) in a thermal cycler GeneAmp 9700 PCR system (Applied Biosystems), according to manufacturer’s recommendations, but reducing the PCR final volume to half (12.5 μl) of the recommended quantity. The electrophoresis was carried out on 3500 Genetic Analyzer (Applied Biosystems, USA) and the fragment analysis was performed with GeneMapper ID-X v.1.4 software (Applied Biosystems). Arlequin version 3.5 [3] was used to calculate allele frequencies, expected heterozygosity (He), observed heterozygosity (Ho), probability value of the HardyWeinberg equilibrium (HWE) exact test. Population genetics statistical parameters were calculated using PowerStats version 1.2.
2. Materials and methods
3. Results
Total of 209 DNA samples was extracted and purified from blood stains or buccal swabs by using the Chelex 100 extraction method [2] from unrelated individuals from Serbia, following informed consent.
Allele frequencies and resulting statistical parameters are given in Table 1. No significant deviations from Hardy–Weinberg equilibrium and linkage disequilibrium were detected within and between the 21
⁎
Corresponding author at: Institute of Forensic Medicine, Clinical Center of Vojvodina, Novi Sad, Serbia. E-mail addresses: [email protected], [email protected] (D. Zgonjanin).
https://doi.org/10.1016/j.fsigss.2019.09.020 Received 10 September 2019; Accepted 21 September 2019 1875-1768/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Dragana Zgonjanin, et al., Forensic Science International: Genetics Supplement Series, https://doi.org/10.1016/j.fsigss.2019.09.020
6 6.2 7 8 9 9.3 10 11 12 13 13.2 14 14.2 14.3 15 15.2 15.3 16 16.2 16.3 17 17.2 17.3 18 18.2 18.3 19 19.2 19.3 20 20.2 20.3 21 21.2 22 22.2 23 23.2 24 24.2 25 25.2 26 26.2 27 27.2 28 28.2 29 29.2 30 30.2 31
Allele/ Locus
0.222
0.278
0.187
0.242
0.268
0.206
0.167
0.019
0.100
0.093
2 0.177 0.005 0.184 0.010 0.144 0.002 0.120
0.105 0.002
0.108 0.002
0.022
FGA
0.208 0.041 0.055
0.196
0.036
0.002
0.251 0.153 0.093 0.196 0.285 0.019 0.002
TH01
0.167
0.002
0.002 0.005
0.043 0.031
0.165 0.038
0.014 0.096 0.232 0.012 0.321 0.038
D19S433
0.005
0.007
0.045
0.268
0.335 0.067 0.053 0.026
0.002 0.196
D2S441
0.053 0.005 0.050 0.002 0.007
0.002
0.010
0.002
0.074
0.112
0.139
0.139 0.002
0.191
0.007 0.005 0.134 0.136
D18S51
0.014
D21S11
0.017
0.017
0.139
0.225
0.055 0.074 0.144 0.323
0.012 0.012
D8S1179
0.033
0.057 0.280 0.017
0.010 0.555 0.079
0.002
TPOX
0.081
0.007
0.033
0.112
0.005
0.007
0.285 0.297 0.325 0.053
CSF1PO
0.067 0.292 0.301 0.148
D16S539
0.002 0.031
vWA
0.014 0.136
D3S1358
Table 1 Allele frequencies and statistical parameters of 21 autosomal STRs loci in a population sample from Serbia (N = 209).
0.148
0.002
0.024
0.100
0.285
0.388
0.148 0.022 0.031
D22S1045
0.007
0.007
0.069 0.333 0.380 0.160
0.043
D5S818
0.026
0.062 0.361 0.268 0.089
0.139 0.055
D13S317
0.007
0.282 0.177 0.129 0.024
0.026 0.175 0.179
D7S820
0.046 0.002
0.046
0.087
0.058
0.043
0.048
0.029
0.048
0.024 0.002 0.007 0.041
0.072 0.010
0.060 0.002
0.077 0.002
0.077
0.063
0.055
0.024 0.002
0.005 0.010
SE33
0.002
0.010
0.029
0.175
0.234
0.266
0.007 0.022 0.256
D10S1248
0.026
0.100 0.002 0.199
0.041 0.141 0.053 0.055 0.100 0.002
0.007
0.012
0.100
0.105
(continued on next page)
0.002
0.017
0.012
0.110
0.065
0.002 0.093
0.017
0.029
0.014 0.134
0.014
0.105
0.146
0.033 0.129
0.060
0.086
0.093
0.234
0.053
0.002
0.065
0.002 0.134
D2S1338
0.005
D12S391
0.072
0.112 0.132 0.081
D1S1656
D. Zgonjanin, et al.
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
0.965 0.785 4.019 0.876
D12S391
0.961 0.785 4.750 0.895
D1S1656
0.019 0.010 0.002 0.005 0.002 0.990 0.785 4.750 0.895
0.019
0.915 0.488 1.900 0.737
D10S1248
0.898 0.472 1.833 0.727 0.862 0.411 1.608 0.689
0.932 0.588 2.430 0.794
4. Discussion In sum, among the 21 markers presents in the commercial kit GlobalFiler™ the most polymorphic, informative and powerful locus in forensic identification was found to be SE33. This marker has the highest PIC, PD, PE, PI and the lowest MP and homozygosity frequency. These results are concordant to others studies, that showed that TPOX is the molecular marker, present in CODIS, that revealed lowest variation among individuals [4].
0.964 0.652 2.903 0.828 0.843 0.434 1.685 0.703
0.887 0.496 1.935 0.742
5. Conclusion
0.898 0.537 2.133 0.766 0.876 0.441 1.713 0.708 0.965 0.717 3.603 0.861 0.941 0.615 2.613 0.809 0.922 0.615 2.613 0.809
0.936 0.571 2.322 0.785
0.914 0.580 2.375 0.790
0.875 0.426 1.659 0.699
0.791 0.245 1.136 0.699
0.931 0.597 2.488 0.799
0.005
0.120 0.007 0.117 0.043
31.2 32 32.2 33.2 34 34.2 35 PD PE PI Ho
This study represents the first report of frequencies for the GlobalFiler™ markers set in a population of Serbia. Power of discrimination, gene diversity and alleles frequencies presented in this study suggest that the 21 STR loci have great value in forensic casework analysis as well as in kinship analysis. PD – power of discrimination, PE – power of exclusion, PI –paternity index, Ho – observed heterozygosity.
SE33 D7S820 D13S317 D5S818 D22S1045 FGA TH01 D19S433 D2S441 D18S51 D21S11 D8S1179 TPOX CSF1PO D16S539 vWA D3S1358 Allele/ Locus
Table 1 (continued)
STR loci. Locus SE33 has shown the greatest power of discrimination in Serbia population, whereas TPOX has was the lowest. The combined power of discrimination was found to be 0.99999999999999999999999989954, whereas the random match probability was found to be 4.2 × 10−26 and the combined power of exclusion was 0.999999990605. Relative to the power of discrimination (PD), the values range between 0.7911 and 0.9899, being the highest and the lowest values given, respectively, by SE33 and TPOX markers. The most informative marker is SE33, which present the highest polymorphic information content (PIC) of 0.941. All of the other markers have PIC values above 0.547. Moreover, the power of exclusion (PE) is 0.2454 for TPOX marker (minimum value) and 0.7847 for SE33 (maximum value). Paternity Index (PI) is situated between 1.1358 (TPOX) and 4.75 (SE33).
0.965 0.597 2.824 0.823
D2S1338
D. Zgonjanin, et al.
Declaration of Competing Interest No conflicts of interest. Acknowledgements The project was supported by Vojvodine Government, by the Grant No. 142-451-2704/2018-01/02 of the Provincial Secretariat for higher education and scientific research of the Vojvodina Province. References [1] J. Buckleton, J. Curran, J. Goudet, D. Taylor, A. Thiery, B.S. Weir, Population-specific FST values for forensic STR markers: a worldwide survey, Forensic Sci. Int. Genet. 23 (2016) 91–100, https://doi.org/10.1016/j.fsigen.2016.03.004. [2] P.S. Walsh, D.A. Metzger, R. Higuchi, Chelex 100 as a medium for simple extraction of DNA for PCR-based typing for forensic material, Biotechniques 10 (1991) 506–513. [3] L. Excoffier, H.E. Lischer, Arlequin suite ver 3. 5: a new series of programs to perform population genetics analyses under Linux and Windows, Mol. Ecol. Resour. 10 (2010) 564–567. [4] P. Thanakiatkrai, T. Kitpipit, Current STR-based techniques in forensic science, Maejo Int. J. Sci. Technol. 7 (2013) 1–15.
3