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Single step strategy for estimating unknown STR allele frequencies in a population
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
Single step strategy for estimating unknown STR allele frequencies in a population Lucia Garrigósa,b, Mariela Caputoa,b, Daniel Coracha,b,* a
Universidad de Buenos Aires - Facultad de Farmacia y Bioquímica, Departamento de Microbiología- Inmunología- Biotecnología y Genética. Centro de Referencia en Identificación Genética Humana, Cátedra de Genética Forense y Servicio de Huellas Digitales Genéticas (SHDG), Junin 956, Ciudad Autónoma de Buenos Aires, 1113, Argentina b Consejo Nacional de Investigaciones Científicas y Técnicas-Conicet, Argentina
ARTICLE INFO
ABSTRACT
Keywords: Allele frequency Single step protocol validation.
Constant growth of forensic DNA databases requires new STR markers to be developed, tested and validated. Aiming to simplify the process of obtaining allele frequency distributions of new STR loci included in new megaplexes, a single step protocol was developed. This goal was achieved by pooling samples of genetically unrelated donors, previously genotyped (n = 99), containing the same amount of DNA (total DNA = 4.5 ng/ul) as measured by fluorometry. Suitability of the method was tested by amplifying commercial kits: GlobalFiler (GF), Verifiler Express (VFE) and Y-filer plus (YFP). Allele peak heights was used to estimate allele frequency and compared with the actual frequency calculated by counting method of the individual pooled samples. The multinomial goodness of fit test was performed for testing statistical differences. In case of VFE, 4.5 ng was the DNA amount that could best estimate the allele frequency distribution; 1 ng for GF and YFP. For simple, complex and compound makers, the highest allele frequencies were correctly estimated (p > 0.05) but in some cases, it was not able to detect alleles in very low frequencies, 2/198 or below, either with VFE or GF. In case of D12S391, either for VFE and GF, some microvariants with frequency of 2, 3 or 5/198 were not assigned although they were present in the profiles, probably due to POP7 resolution. In case of YFP, some alleles were not detected at DYS385. Nevertheless, with some limitations, the proposed strategy proved to be fast and efficient for major allele frequency estimation. This simple approach generates an empirical distribution that allows for the estimation of population allele frequencies in a single step assay.
1. Introduction
2. Materials & methods
DNA-based genotyping has been used successfully since the early 90’s and is the gold standard for assisting in personal identification [1]. Continuous growth of forensic DNA databases requires new STR markers to be developed, tested and validated [2]. Aiming to simplify the process of obtaining allele frequency distributions of new STR loci included in new megaplexes, a single step protocol was developed.
We designed a protocol that included: a selection step of previously typed samples belonging to unrelated donors, a careful quantification of DNA concentrations and a pooling process (n = 99), containing the same DNA concentration (total DNA = 4.5 ng/ul) as measured by fluorometry. Suitability of the method was tested by amplifying commercial kits: GlobalFiler (GF) [3], Verifiler Express (VFE) [4] and Y-filer
⁎
Corresponding author: Junin 956, Piso 7, 1113 Buenos Aires, Argentina. E-mail address: [email protected] (D. Corach).
https://doi.org/10.1016/j.fsigss.2019.10.155 Received 11 September 2019; Received in revised form 11 October 2019; Accepted 11 October 2019 1875-1768/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Lucia Garrigós, Mariela Caputo and Daniel Corach, Forensic Science International: Genetics Supplement Series, https://doi.org/10.1016/j.fsigss.2019.10.155
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
L. Garrigós, et al.
Fig. 1. Example of markers amplified by VeriFiler Express (left) and graphic showing the frequency estimation from allele peaks height compared with the actual frequency calculated by counting method (right). It is shown with orange arrow the alleles with a frequency < 2/198 and with green arrow the alleles with a frequency between 2/198 and 5/198.
plus (YFP) [5], (Applied Biosystems, Foster City, USA). Amplicon detection was carried out in an ABI-3500 using POP-7 and a 50-cm capillary array. Allele peaks height were used to estimate allele frequency and compared with the actual frequency calculated by counting of the individual pooled samples. The multinomial goodness of fit test was performed for testing statistical differences [6].
system were detected although they were not present at the pool samples probably due to an excess of stutter from the real allele. Despite these little discrepancies, no statistical difference were obtained between the estimation and the actual allele frequencies of the tested systems.
3. Results
4. Conclusions
In case of VFE, 4.5 ng was the DNA amount that could best estimate the allele frequency distribution; 1 ng for GF and YFP according to the internal laboratory validation. Analysis method used was for simple, complex and compound makers, the highest allele frequencies were correctly estimated (p > 0.05). In some cases, it was not able to detect alleles in very low frequencies 2/198 or 1/198 either with VFE or GF. In the case of D12S391and D1S1656 for VFE and GF, some microvariants with frequencies of 2, 3 or 5/198 were not assigned although they were present in the profiles, probably due to poor POP7 resolution rather than the pooling strategy. See Fig. 1 and 2 for details. In the case of YFP, only the allele 18 from DYS385 was not detected (frequency < 2/56) (data not shown). Alleles 5 from TH0-1, 13 from SE33 and 11 from D18S51 at GF system; 15.2 and 18.1 from D18S51 at VFE
The frequency estimation was not influenced by the marker structure (simple, complex or compound locus). The highest alleles frequencies were correctly estimated in all the tested systems. Alleles with frequencies below 2/198 were not detected with the amplification and analysis conditions (internal lab validation for routine samples). Some alleles with frequencies between 2/198 and 5/198 were not assigned by the analysis method but this could be attributed to the POP7 resolution rather than the proposed strategy. Alleles with frequencies higher the minimal frequency (5/198 = 0,025) were detected in all cases. Finally, despite some limitations, the proposed strategy proved to be fast and efficient for the majority of the allele frequency estimations. This simple approach generates an empirical distribution that can estimate allele frequencies in a population via a single step assay.
2
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
L. Garrigós, et al.
Fig. 2. Example of markers amplified by GlobalFiler (left) and graphic showing the frequency estimation from allele peaks height compared with the actual frequency calculated by counting method (right). It is shown with orange arrow the alleles with a frequency < 2/198 and with green arrow the alleles with a frequency between 2/198 and 5/198.
Conflict of interest
References
The authors report no declarations of interest.
[1] R. Thompson, S. Zoppis, B. McCord, An overview of DNA typing methods for human identification: past, present, and future, Methods Mol. Biol. 830 (2012) 3–16. [2] Butler, J.M., The future of forensic DNA analysis, Philos Trans R Soc Lond B Biol Sci .2-15:370. [3] VeriFiler™ Plus PCR Amplification. Publication Number MAN001743. Revision B.0. [4] GlobalFiler™ PCR Amplification Kit USER GUIDE. Revision E. Publication Number 4477604. [5] Yfiler® Plus PCR Amplification Kit. Publication Number 4485610.Revision B. [6] Xlstat by addinsoft.
Acknowledgments The research was supported by funds rised as genuine resources by SHDG and 20020170100721BA (UBACyT) to Daniel Corach.
3
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/fsigss
Single step strategy for estimating unknown STR allele frequencies in a population Lucia Garrigósa,b, Mariela Caputoa,b, Daniel Coracha,b,* a
Universidad de Buenos Aires - Facultad de Farmacia y Bioquímica, Departamento de Microbiología- Inmunología- Biotecnología y Genética. Centro de Referencia en Identificación Genética Humana, Cátedra de Genética Forense y Servicio de Huellas Digitales Genéticas (SHDG), Junin 956, Ciudad Autónoma de Buenos Aires, 1113, Argentina b Consejo Nacional de Investigaciones Científicas y Técnicas-Conicet, Argentina
ARTICLE INFO
ABSTRACT
Keywords: Allele frequency Single step protocol validation.
Constant growth of forensic DNA databases requires new STR markers to be developed, tested and validated. Aiming to simplify the process of obtaining allele frequency distributions of new STR loci included in new megaplexes, a single step protocol was developed. This goal was achieved by pooling samples of genetically unrelated donors, previously genotyped (n = 99), containing the same amount of DNA (total DNA = 4.5 ng/ul) as measured by fluorometry. Suitability of the method was tested by amplifying commercial kits: GlobalFiler (GF), Verifiler Express (VFE) and Y-filer plus (YFP). Allele peak heights was used to estimate allele frequency and compared with the actual frequency calculated by counting method of the individual pooled samples. The multinomial goodness of fit test was performed for testing statistical differences. In case of VFE, 4.5 ng was the DNA amount that could best estimate the allele frequency distribution; 1 ng for GF and YFP. For simple, complex and compound makers, the highest allele frequencies were correctly estimated (p > 0.05) but in some cases, it was not able to detect alleles in very low frequencies, 2/198 or below, either with VFE or GF. In case of D12S391, either for VFE and GF, some microvariants with frequency of 2, 3 or 5/198 were not assigned although they were present in the profiles, probably due to POP7 resolution. In case of YFP, some alleles were not detected at DYS385. Nevertheless, with some limitations, the proposed strategy proved to be fast and efficient for major allele frequency estimation. This simple approach generates an empirical distribution that allows for the estimation of population allele frequencies in a single step assay.
1. Introduction
2. Materials & methods
DNA-based genotyping has been used successfully since the early 90’s and is the gold standard for assisting in personal identification [1]. Continuous growth of forensic DNA databases requires new STR markers to be developed, tested and validated [2]. Aiming to simplify the process of obtaining allele frequency distributions of new STR loci included in new megaplexes, a single step protocol was developed.
We designed a protocol that included: a selection step of previously typed samples belonging to unrelated donors, a careful quantification of DNA concentrations and a pooling process (n = 99), containing the same DNA concentration (total DNA = 4.5 ng/ul) as measured by fluorometry. Suitability of the method was tested by amplifying commercial kits: GlobalFiler (GF) [3], Verifiler Express (VFE) [4] and Y-filer
⁎
Corresponding author: Junin 956, Piso 7, 1113 Buenos Aires, Argentina. E-mail address: [email protected] (D. Corach).
https://doi.org/10.1016/j.fsigss.2019.10.155 Received 11 September 2019; Received in revised form 11 October 2019; Accepted 11 October 2019 1875-1768/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Lucia Garrigós, Mariela Caputo and Daniel Corach, Forensic Science International: Genetics Supplement Series, https://doi.org/10.1016/j.fsigss.2019.10.155
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
L. Garrigós, et al.
Fig. 1. Example of markers amplified by VeriFiler Express (left) and graphic showing the frequency estimation from allele peaks height compared with the actual frequency calculated by counting method (right). It is shown with orange arrow the alleles with a frequency < 2/198 and with green arrow the alleles with a frequency between 2/198 and 5/198.
plus (YFP) [5], (Applied Biosystems, Foster City, USA). Amplicon detection was carried out in an ABI-3500 using POP-7 and a 50-cm capillary array. Allele peaks height were used to estimate allele frequency and compared with the actual frequency calculated by counting of the individual pooled samples. The multinomial goodness of fit test was performed for testing statistical differences [6].
system were detected although they were not present at the pool samples probably due to an excess of stutter from the real allele. Despite these little discrepancies, no statistical difference were obtained between the estimation and the actual allele frequencies of the tested systems.
3. Results
4. Conclusions
In case of VFE, 4.5 ng was the DNA amount that could best estimate the allele frequency distribution; 1 ng for GF and YFP according to the internal laboratory validation. Analysis method used was for simple, complex and compound makers, the highest allele frequencies were correctly estimated (p > 0.05). In some cases, it was not able to detect alleles in very low frequencies 2/198 or 1/198 either with VFE or GF. In the case of D12S391and D1S1656 for VFE and GF, some microvariants with frequencies of 2, 3 or 5/198 were not assigned although they were present in the profiles, probably due to poor POP7 resolution rather than the pooling strategy. See Fig. 1 and 2 for details. In the case of YFP, only the allele 18 from DYS385 was not detected (frequency < 2/56) (data not shown). Alleles 5 from TH0-1, 13 from SE33 and 11 from D18S51 at GF system; 15.2 and 18.1 from D18S51 at VFE
The frequency estimation was not influenced by the marker structure (simple, complex or compound locus). The highest alleles frequencies were correctly estimated in all the tested systems. Alleles with frequencies below 2/198 were not detected with the amplification and analysis conditions (internal lab validation for routine samples). Some alleles with frequencies between 2/198 and 5/198 were not assigned by the analysis method but this could be attributed to the POP7 resolution rather than the proposed strategy. Alleles with frequencies higher the minimal frequency (5/198 = 0,025) were detected in all cases. Finally, despite some limitations, the proposed strategy proved to be fast and efficient for the majority of the allele frequency estimations. This simple approach generates an empirical distribution that can estimate allele frequencies in a population via a single step assay.
2
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
L. Garrigós, et al.
Fig. 2. Example of markers amplified by GlobalFiler (left) and graphic showing the frequency estimation from allele peaks height compared with the actual frequency calculated by counting method (right). It is shown with orange arrow the alleles with a frequency < 2/198 and with green arrow the alleles with a frequency between 2/198 and 5/198.
Conflict of interest
References
The authors report no declarations of interest.
[1] R. Thompson, S. Zoppis, B. McCord, An overview of DNA typing methods for human identification: past, present, and future, Methods Mol. Biol. 830 (2012) 3–16. [2] Butler, J.M., The future of forensic DNA analysis, Philos Trans R Soc Lond B Biol Sci .2-15:370. [3] VeriFiler™ Plus PCR Amplification. Publication Number MAN001743. Revision B.0. [4] GlobalFiler™ PCR Amplification Kit USER GUIDE. Revision E. Publication Number 4477604. [5] Yfiler® Plus PCR Amplification Kit. Publication Number 4485610.Revision B. [6] Xlstat by addinsoft.
Acknowledgments The research was supported by funds rised as genuine resources by SHDG and 20020170100721BA (UBACyT) to Daniel Corach.
3