- Email: [email protected]
IdentiPLEX: A new STR kit for human identification developed and validated at IdentiGEN laboratory in Colombia
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
IdentiPLEX: A new STR kit for human identification developed and validated at IdentiGEN laboratory in Colombia J.D. Grandaa, O. Palacioa, C.G. Freyle-Baróna, K.M. Maldonado-Uquillasb, A. Ibarraa, G. Burgosc,* a
IdentiGEN - Genetic Identification Laboratory and Research Group of Genetic Identification, Institute of Biology, School of Natural and Exact Sciences (FCEN), University of Antioquia, Medellín, Colombia b Carrera de Biotecnología, Facultad de Ingeniería y Ciencias Aplicadas, Universidad de Las Américas (UDLA), Quito, Ecuador c Escuela de Medicina, Facultad de Ciencias de la Salud, Universidad de Las Américas (UDLA), Quito, Ecuador
ARTICLE INFO
ABSTRACT
Keywords: STR molecular markers Paternity testing kit
Colombian laws require that paternity testing probability (W) values be higher than 99,99% to prove biological relationship. IdentiGEN lab currently uses 23 autosomal genetic markers included in the commercial kits Identifiler®, PowerPlex®16, FFFL® and Triplex® (in-house kit: D18S535, D2S1338, SE33); however, in some complex cases the number of markers is not enough to reach the legal statistical threshold. IdentiPLEX kit, consisting of 19 markers, selected from NIST & STRbase databases was developed and validated according to the requirements established by the norm NTC-ISO17025. These include among others, studies of precision, specificity, sensitivity, stutter proportion and concordance tests. Forensic and population parameters were also calculated using Powerstats and Arlequin V3.5.2 software (N = 701 individuals). These analyses allowed to conclude that identiplex kit is appropriate for routine analysis casework, and more specifically aiming to solve criminal and filiation complex cases.
1. Introduction DNA test is the gold standard in the solution of criminal cases and civil proceedings. Genetic identification is a legal requirement to determine the identity of the suspect/victim (acquires a preponderant role due to the situation of armed violence in Colombia), and allows to discard/affirm biological relationships in cases of filiation. Colombian Law No. 721 demands that probability of paternity (W) must overcome 99.99% [1]. Nowadays, to solve paternity tests the IdentiGEN lab at University of Antioquia works with 23 markers type autosomal STR included in the kits Identifiler® (Applied Biosystems), PowerPlex 16®, FFFL® (Promega Co.) and Triplex (in-house kit: D18S535, D2S1338, SE33). In some complex cases, when the statistical threshold is not reached with the use of a single kit, typing with the others is necessary to increase the number of markers in order to exceed the threshold required by law, which results in higher costs and times. In the present study, a new in-house kit called IndentiPLEX was designed, developed and validated. This kit picks up 18 autosomal STRs markers (D5S818, D13S317, D7S820, D16S539, PENTA E, VWA, D12S391, PENTA D, CSF1PO, D1S1656, TH01, SE33, F13B, D21S11, D18S51, D10S1248, D22S1045, D2S441 plus Amelogenin) in order to optimize costs and time for genotyping of samples in routine and complex casework. This kit was validated as required by the National Accreditation Agency of
⁎
Colombia (ONAC) and the technical standard NTC-ISO/IEC 17025 [2]. The IdentiPLEX kit allows the analysis for simple and complex cases of filiation, as well as for forensic cases. Validation yielded satisfactory results of performance, concordance, reproducibility, and specificity for human DNA. Its use reduced costs for reagents, increased the number of STR markers and optimized the lab’s workflow. 2. Methods Criteria for selection of STR genetic markers were: 1.-All them are used and are recognized by international forensic community. 2.-Their sequences are disclosed in the forensic scientific community. 3.-Markers are included in commercial kits and can be used in the interlaboratory exercises of the Spanish and Portuguese Speaking Group of the International Society for Forensic Genetics (GHEP-ISFG). Sequences for STRs and mini-STRs primers were obtained from databases available on the web pages NIST Standard Reference Data and STRbase (Table S1) [3]. Primers were labeled with fluorochromes (FAM, VIC, NED, PET), included in the DS-33 matrix that use G5 filter of Applied Biosystems chemistry. PCR conditions were optimized, and their efficiency was evaluated with DNA extracted by Chelex or FTA cards method, from blood (FTA and DMA cards), oral mucosa (FTA card and cotton swab) and amniotic fluid (FTA card) samples. Amplicons were mixed with LIZ
Corresponding author. E-mail address: [email protected] (G. Burgos).
https://doi.org/10.1016/j.fsigss.2019.10.083 Received 15 September 2019; Accepted 7 October 2019 1875-1768/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: J.D. Granda, et al., Forensic Science International: Genetics Supplement Series, https://doi.org/10.1016/j.fsigss.2019.10.083
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
J.D. Granda, et al.
Fig. 1. Typical electropherogram of human DNA amplified with IdentiPLEX kit (blood spoted on FTA card).
managed to solve 6 of 21 inconclusive cases with probabilities higher than 99.99% (Table S3).
500 and were separated by capillary electrophoresis in the ABI 3130 genetic analyzer as usual conditions. An in-house allelic ladder was constructed and used as size control for automatic allelic assignment performed in GeneMapper® ID V3.2 software (Applied Biosystems). Tests of precision, specificity, sensitivity, concordance studies and stutter rate estimation were executed for IdentiPLEX validation. To evaluate specificity DNA samples of animals and plants such as Gallus gallus, Canis lupus familiaris, Anthurium andreanum, Anopheles sp., Bos taurus and Equus caballus were tested. For the population genetic data analysis, 701 blood samples (FTA cards) were used from individuals born in the province of Antioquia, not biologically related, that signed informed consent. Allelic and genotypic frequencies were calculated by direct counting. The Arlequin software V3.5.2 was used to evaluate the genetic diversity, expected and observed heterozygosity, Hardy-Weinberg equilibrium and linkage equilibrium analysis, using Bonferroni transformed p-values. PowerStats (Promega Co.) [4] was used to calculate forensic and paternity parameters. IdentiPLEX efficiency was evaluated, processing inconclusive cases between 2011–2015, previously analyzed with routine autosomal STRs kits.
4. Conclusion The IdentiPLEX kit reduced costs and processing time for STRs genotyping. Results obtained in this study were accurate, precise, reproducible and reliable; therefore, using this approach could solve routine and complex caseworks, improving workflow in forensic labs. Role of funding This work was funded by the IdentiGEN lab Medellín-Colombia, GB & KM were supported by grant MED.GB.18.06 from the Dirección General de Investigación at the Universidad de Las Américas Quito-Ecuador. Ethical standards The use of biological samples was authorized by signing the informed consent for population studies, approved by the Bioethics Committee of the Faculty of Medicine of the University of Antioquia (Medellín-Colombia).
3. Results and discussion The kit IdentiPLEX allows to solve paternity cases from blood, saliva and amniotic fluid samples, stored in different supports. The samples evaluated with the IdentiPLEX kit presented complete and balanced profiles, intermediate precision and high repproducibility that permits obtaining uniform and exact measurements (Fig. 1). In specificity evaluation, no amplicons were obtained with non-human DNA. In sensibility studies, DNA 2800 M positive control (Promega® Co.) showed complete genetic profiles with DNA equal to or greater than 0.5 ng and the stutter rate of the genetic markers ranges from 3.8% to 15.68%. The genetic profiles generated by IdentiPLEX showed 100% concordance with the biological samples belonging to the GHEP-ISFG interlaboratory tests (Data available upon request). According to population genetic tests, the STRs molecular markers included in IdentiPLEX kit were in Hardy-Weinberg equilibrium (Table S2). Markers did not present a linkage imbalance between them after Bonferroni (α = 0.05/18 = 0.0027). Finally, the new kit was used to reanalyze 21 complex cases whose probability of biological relationship had not previously reached the limits established by law; IdentiPLEX kit
Declaration of Competing Interest None. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.fsigss.2019.10.083. References [1] Republica de Colombia. Congreso de la República de Colombia. 2001. Ley 721 de 2001 (diciembre 24). Por medio de la cual se modifica la Ley 75 de 1968. [2] Instituto Colombiano de Normas Técnicas y Certificación, IEC-17025 “Requisitos generales para la competencia de los laboratorios de ensayo y calibración”, Oficina Nacional de Normalización, 2005. [3] STRBase, (2014) http://www.cstl.nist.gov/strbase/. [4] Promega Corporation, PowerStats Computer Program for the Analysis of Population Statistics, (1999) http://www.promega.com/geneticidtools.
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Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/fsigss
IdentiPLEX: A new STR kit for human identification developed and validated at IdentiGEN laboratory in Colombia J.D. Grandaa, O. Palacioa, C.G. Freyle-Baróna, K.M. Maldonado-Uquillasb, A. Ibarraa, G. Burgosc,* a
IdentiGEN - Genetic Identification Laboratory and Research Group of Genetic Identification, Institute of Biology, School of Natural and Exact Sciences (FCEN), University of Antioquia, Medellín, Colombia b Carrera de Biotecnología, Facultad de Ingeniería y Ciencias Aplicadas, Universidad de Las Américas (UDLA), Quito, Ecuador c Escuela de Medicina, Facultad de Ciencias de la Salud, Universidad de Las Américas (UDLA), Quito, Ecuador
ARTICLE INFO
ABSTRACT
Keywords: STR molecular markers Paternity testing kit
Colombian laws require that paternity testing probability (W) values be higher than 99,99% to prove biological relationship. IdentiGEN lab currently uses 23 autosomal genetic markers included in the commercial kits Identifiler®, PowerPlex®16, FFFL® and Triplex® (in-house kit: D18S535, D2S1338, SE33); however, in some complex cases the number of markers is not enough to reach the legal statistical threshold. IdentiPLEX kit, consisting of 19 markers, selected from NIST & STRbase databases was developed and validated according to the requirements established by the norm NTC-ISO17025. These include among others, studies of precision, specificity, sensitivity, stutter proportion and concordance tests. Forensic and population parameters were also calculated using Powerstats and Arlequin V3.5.2 software (N = 701 individuals). These analyses allowed to conclude that identiplex kit is appropriate for routine analysis casework, and more specifically aiming to solve criminal and filiation complex cases.
1. Introduction DNA test is the gold standard in the solution of criminal cases and civil proceedings. Genetic identification is a legal requirement to determine the identity of the suspect/victim (acquires a preponderant role due to the situation of armed violence in Colombia), and allows to discard/affirm biological relationships in cases of filiation. Colombian Law No. 721 demands that probability of paternity (W) must overcome 99.99% [1]. Nowadays, to solve paternity tests the IdentiGEN lab at University of Antioquia works with 23 markers type autosomal STR included in the kits Identifiler® (Applied Biosystems), PowerPlex 16®, FFFL® (Promega Co.) and Triplex (in-house kit: D18S535, D2S1338, SE33). In some complex cases, when the statistical threshold is not reached with the use of a single kit, typing with the others is necessary to increase the number of markers in order to exceed the threshold required by law, which results in higher costs and times. In the present study, a new in-house kit called IndentiPLEX was designed, developed and validated. This kit picks up 18 autosomal STRs markers (D5S818, D13S317, D7S820, D16S539, PENTA E, VWA, D12S391, PENTA D, CSF1PO, D1S1656, TH01, SE33, F13B, D21S11, D18S51, D10S1248, D22S1045, D2S441 plus Amelogenin) in order to optimize costs and time for genotyping of samples in routine and complex casework. This kit was validated as required by the National Accreditation Agency of
⁎
Colombia (ONAC) and the technical standard NTC-ISO/IEC 17025 [2]. The IdentiPLEX kit allows the analysis for simple and complex cases of filiation, as well as for forensic cases. Validation yielded satisfactory results of performance, concordance, reproducibility, and specificity for human DNA. Its use reduced costs for reagents, increased the number of STR markers and optimized the lab’s workflow. 2. Methods Criteria for selection of STR genetic markers were: 1.-All them are used and are recognized by international forensic community. 2.-Their sequences are disclosed in the forensic scientific community. 3.-Markers are included in commercial kits and can be used in the interlaboratory exercises of the Spanish and Portuguese Speaking Group of the International Society for Forensic Genetics (GHEP-ISFG). Sequences for STRs and mini-STRs primers were obtained from databases available on the web pages NIST Standard Reference Data and STRbase (Table S1) [3]. Primers were labeled with fluorochromes (FAM, VIC, NED, PET), included in the DS-33 matrix that use G5 filter of Applied Biosystems chemistry. PCR conditions were optimized, and their efficiency was evaluated with DNA extracted by Chelex or FTA cards method, from blood (FTA and DMA cards), oral mucosa (FTA card and cotton swab) and amniotic fluid (FTA card) samples. Amplicons were mixed with LIZ
Corresponding author. E-mail address: [email protected] (G. Burgos).
https://doi.org/10.1016/j.fsigss.2019.10.083 Received 15 September 2019; Accepted 7 October 2019 1875-1768/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: J.D. Granda, et al., Forensic Science International: Genetics Supplement Series, https://doi.org/10.1016/j.fsigss.2019.10.083
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
J.D. Granda, et al.
Fig. 1. Typical electropherogram of human DNA amplified with IdentiPLEX kit (blood spoted on FTA card).
managed to solve 6 of 21 inconclusive cases with probabilities higher than 99.99% (Table S3).
500 and were separated by capillary electrophoresis in the ABI 3130 genetic analyzer as usual conditions. An in-house allelic ladder was constructed and used as size control for automatic allelic assignment performed in GeneMapper® ID V3.2 software (Applied Biosystems). Tests of precision, specificity, sensitivity, concordance studies and stutter rate estimation were executed for IdentiPLEX validation. To evaluate specificity DNA samples of animals and plants such as Gallus gallus, Canis lupus familiaris, Anthurium andreanum, Anopheles sp., Bos taurus and Equus caballus were tested. For the population genetic data analysis, 701 blood samples (FTA cards) were used from individuals born in the province of Antioquia, not biologically related, that signed informed consent. Allelic and genotypic frequencies were calculated by direct counting. The Arlequin software V3.5.2 was used to evaluate the genetic diversity, expected and observed heterozygosity, Hardy-Weinberg equilibrium and linkage equilibrium analysis, using Bonferroni transformed p-values. PowerStats (Promega Co.) [4] was used to calculate forensic and paternity parameters. IdentiPLEX efficiency was evaluated, processing inconclusive cases between 2011–2015, previously analyzed with routine autosomal STRs kits.
4. Conclusion The IdentiPLEX kit reduced costs and processing time for STRs genotyping. Results obtained in this study were accurate, precise, reproducible and reliable; therefore, using this approach could solve routine and complex caseworks, improving workflow in forensic labs. Role of funding This work was funded by the IdentiGEN lab Medellín-Colombia, GB & KM were supported by grant MED.GB.18.06 from the Dirección General de Investigación at the Universidad de Las Américas Quito-Ecuador. Ethical standards The use of biological samples was authorized by signing the informed consent for population studies, approved by the Bioethics Committee of the Faculty of Medicine of the University of Antioquia (Medellín-Colombia).
3. Results and discussion The kit IdentiPLEX allows to solve paternity cases from blood, saliva and amniotic fluid samples, stored in different supports. The samples evaluated with the IdentiPLEX kit presented complete and balanced profiles, intermediate precision and high repproducibility that permits obtaining uniform and exact measurements (Fig. 1). In specificity evaluation, no amplicons were obtained with non-human DNA. In sensibility studies, DNA 2800 M positive control (Promega® Co.) showed complete genetic profiles with DNA equal to or greater than 0.5 ng and the stutter rate of the genetic markers ranges from 3.8% to 15.68%. The genetic profiles generated by IdentiPLEX showed 100% concordance with the biological samples belonging to the GHEP-ISFG interlaboratory tests (Data available upon request). According to population genetic tests, the STRs molecular markers included in IdentiPLEX kit were in Hardy-Weinberg equilibrium (Table S2). Markers did not present a linkage imbalance between them after Bonferroni (α = 0.05/18 = 0.0027). Finally, the new kit was used to reanalyze 21 complex cases whose probability of biological relationship had not previously reached the limits established by law; IdentiPLEX kit
Declaration of Competing Interest None. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.fsigss.2019.10.083. References [1] Republica de Colombia. Congreso de la República de Colombia. 2001. Ley 721 de 2001 (diciembre 24). Por medio de la cual se modifica la Ley 75 de 1968. [2] Instituto Colombiano de Normas Técnicas y Certificación, IEC-17025 “Requisitos generales para la competencia de los laboratorios de ensayo y calibración”, Oficina Nacional de Normalización, 2005. [3] STRBase, (2014) http://www.cstl.nist.gov/strbase/. [4] Promega Corporation, PowerStats Computer Program for the Analysis of Population Statistics, (1999) http://www.promega.com/geneticidtools.
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