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Bone sampling criteria for DNA genotyping: macroscopic sample categorization and STR typing results
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
Bone sampling criteria for DNA genotyping: macroscopic sample categorization and STR typing results Carlos Vullo*, Andrea Rocha, Carola Romanini, Magdalena Romero, Laura Catelli, Martina Rotondo, Micaela Longaray EAAF Forensic Genetic Lab, Independencia, 644-3A, Cordoba, Argentina
A R T I C LE I N FO
A B S T R A C T
Keywords: Bone sampling Macroscopic bone aspect DNA yield STR typing results
The Argentine Forensic Anthropology Team (EAAF) investigates human rights violations which took place in Argentina during the military government that ruled out from 1976 to 1983 and also cooperates in other countries with humanitarian crisis as well. Most of the bone remains that the EAAF analyses show long post mortem interval. Consequently, the skeletal remains may have been exposed to different conditions such as moisture, temperature and pH conditions, microorganisms and different types of soil that can produce various degrees of preservation. On the other hand, severely burnt remains can make the genetic analysis more complex. All the above mentioned influences the DNA quantity and quality. In the present study, we scored the samples to investigate the correlation between the bone macroscopic aspect and the concentration of the DNA extracted from it. Samples were classified into four major groups: score 1 was assigned to samples with very good preservation, score 2 to those showing slight superficial alterations. Score 3 and 4 samples showed regular and bad macroscopic appearance, respectively. Our results clearly supports that samples with good preservation show high well-preserved DNA concentrations and also a higher number of reportable STR markers in comparison to those ranked in the worst categories. This work proposes a useful guide that can help anthropologists to make a more efficient selection of aged-samples for genetic studies.
1. Introduction The main problems of DNA extraction from skeletal material are DNA degradation due to environmental factors and the presence of soilderived inhibitors like humic acids. The quality of DNA preservation in these samples is highly variable and often limited and/or degraded. Bone and teeth clearly protect DNA from environmental degradation and biological attack through their physical and chemical robustness. It seems reasonable to suppose that the characteristics of skeletal remains that are correlated with their long term survival would be those that contribute to the protection of DNA from degradation. Because of this, it is expected that samples with a good state of preservation will lead to high quantity and quality DNA recovery in comparison to those samples with a poor visual aspect. The aim of this study is to evaluate the macroscopic aspect of different types of skeletal remains and to compare it with STR typing results. The efficiency of macroscopic classification was determined on the basis of bone sample DNA concentration obtained by Real-Time PCR assay and the 15 loci from any of the STR amplification kits used in
⁎
this analysis. These data can be a useful guide for sample selection of skeletal remains with a greater chance of obtaining good results based on their macroscopic appearance. 2. Materials and methods A total of 1727 skeletal remains were analyzed, including teeth and 19 different types of bone pieces. Most of the processed samples have over 35 years of post-mortem interval. Based on our experience, the samples were classified into four categories according to the visual macroscopic aspect of skeletal samples usually received by our laboratory. Totally burned samples were not included because the reproducibility of genetic results is extremely difficult. Score 1 corresponds to samples with very good appearance: bones in this category have few spots and / or pigmentations that can be easily removed or they may not even be present at all. They have a strong firmness and abundant cortical portion. Teeth keep their structure intact without the presence of caries and they may also have a slight coloration.
Corresponding author. E-mail address: [email protected] (C. Vullo).
https://doi.org/10.1016/j.fsigss.2019.10.167 Received 9 September 2019; Accepted 16 October 2019 1875-1768/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Carlos Vullo, et al., Forensic Science International: Genetics Supplement Series, https://doi.org/10.1016/j.fsigss.2019.10.167
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
C. Vullo, et al.
Score 2 involves samples with a good state of preservation: teeth may have some superficial caries and / or spots that are completely removed during the cleaning, their structure remains intact. Bones can also show superficial alterations that are totally removed during the cleaning. The cortical portion is still generous. Score 3 applies to regular visual appearance samples: bones show marked alterations in their surface and their structure is compromised. The cortical fraction decreases significantly and the sample adopts a sandy appearance that contributes to a shedding of abundant dust particles during the cleaning and, therefore, a large quantity of material is lost. Teeth samples may present deep caries with highly dark spots; and the roots, in most of the cases, present open ends. During the cleaning process of this type of samples there is no great improvement in their appearance since the mentioned alterations persist. Score 4 includes pieces with a poor state of preservation: bones have a fragile-sandy appearance and even scales can be observed due to the alteration of their structure. The cortical portion is practically absent after cleaning. Teeth can be compromised due to the abundant holes in their structure, numerous spots, fractures and one or more roots broken leaving their interior exposed. Samples in this category are extremely fragile and tend to break while they are being cleaned or at the slightest contact with any other material. Superficial alterations include the presence of fungi, coloration due to the type of soil and/or presence of crystals that may have also been on the ground, as well as perforations due to termites, among others. Fig. 1. Examples of bones and teeth for all the proposed scores (above) and average DNA concentration and reportable STR for each score.
2.1. Sample preparation All samples were mechanically cleaned, washed, exposed to 10% sodium hypochlorite for 10 min and washed again; finally briefly rinsed with 96% ethanol and irradiated with UV-light (254 nm) for 15 min per side. Samples were pulverized using 6770 Freezer Mill (SPEX CertiPrep, Metuchen, NJ, USA) [1].
3. Results and discussion Real-Time PCR quantification revealed that samples with a very good and good visual appearance (scores 1 and 2) have higher DNA concentration values than those with a regular or bad appearance (scores 3 and 4) regardless of the extraction method used. On the other hand, it can be observed that the average number of genetic markers reported is directly related to DNA concentration [7] (Fig. 1). Low DNA concentration obtained for regular and bad appearance samples can be explained by the fact that their structure is not preserved and therefore, DNA is totally exposed to severe environmental conditions. In contrast, skeletal remains with very good and good macroscopic aspect presented a considerably higher DNA concentration as their structure still maintains all the characteristics that protect the DNA from external agents. Therefore, these samples have less degraded DNA than those with opposite characteristics. The highest DNA recovery was observed on bones with dense cortical fraction, intact structure and no several superficial alterations. The same results were found for teeth that had all their roots without open end or major caries and/or spots. A significant correlation (p < 0.05; Tukey-Kramer Multiple Comparison Test) between the described scores was found.
2.2. DNA extraction methods This study extends over several years of work and our laboratory has used two different extraction methods during that period: A) Qiagen Blood Maxi Kit (Qiagen, Valencia, CA, USA) and B) PrepFiler BTA Forensic Extraction Kit (Applied Biosystems, Foster City, CA, USA) on Automate Express platform. In both protocols the amount of starting material was increased. For extraction method A, samples were previously demineralized with 0.5 M EDTA pH 8 at room temperature for 72 h with rotation movements [2,3]. For method B, bone powder was treated with lysis buffer (0.5 M EDTA and 1% N-Laurylsarcosinate), proteinase K 20 mg/mL (Invitrogen, Carlsbad, CA, USA) and 1.0 M DTT (Sigma-Aldrich) and incubated overnight at 56 °C for full demineralization [4–6]. 2.3. DNA quantification and amplification
4. Conclusions Human genomic DNA was determined by using the Quantifiler™ Human DNA Quantification Kit or Quantifiler™ Human Plus DNA Quantification Kit (Applied Biosystems, Foster City, CA, USA) and 7500 Real-Time PCR System (Applied Biosystems). In order to compare the data obtained with both kits, the information of the smaller amplicon of the autosomal target for Quantifiler™ Human plus DNA Quantification Kit was taken into account. PCR amplification was carried out by using AmpFLSTR™ Identifiler™ Plus PCR Amplification Kit, GlobalFiler™ PCR Amplification Kit (Applied Biosystems, Foster City, CA, USA) and PowerPlex® Fusion System (Promega, Madison, WI, USA). Only STR markers shared by these kits were considered in the present study. Amplified products were separated and detected on ABI PRISM 3130 Genetic Analyzer or 3500 Genetic Analyzer (Applied Biosystems).
Our results show a significant correlation between described scores based on the macroscopic appearance of bone samples with DNA recovery and the number of reportable STRs. This work proposes a guide useful for anthropologists to make a more efficient sample selection of aged-samples, contributing to DNA typing successs. References [1] W. Goodwin, Forensic DNA Typing Protocols, second edition, Humana Press, New York, 2016. [2] J. Davoren, D. Vanek, R. Konjhodzić, et al., Highly effective DNA extraction method for nuclear short tandem repeat testing of skeletal remains from mass graves, Croat. Med. J. 48 (4) (2007) 478-85. [3] C. Romanini, M.L. Catelli, A. Borosky, et al., Typing short amplicon binary
2
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
C. Vullo, et al.
London, 2011. [6] O. Loreille, T.M. Diegoli, J.A. Irwin, M.D. Coble, T.J. Parson, High efficiency DNA extraction bone by total demineralization, Forensic Sci. Int. Genet. 1 (2006) 191-5. [7] A. Milos, A. Semanovic, L. Smajlovic, et al., Success rates of nuclear STR typing from different skeletal elements, Croat. Med. J. 48 (2007) 486-93.
polymorphisms: supplementary SNP and Indel genetic information in the analysis of highly degraded skeletal remains, Forensic Sci. Int. 6 (4) (2012) 469–476. [4] S. Amory, R. Huel, A. Bilic´, et al., Automatable full demineralization DNA extraction procedure from degraded skeletal remains, Forensic Sci. Int. Genet. 6 (3) (2012) 398–406. [5] J.M. Butler, Advanced Topics in Forensic DNA Typing: Methodology, Academic,
3
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/fsigss
Bone sampling criteria for DNA genotyping: macroscopic sample categorization and STR typing results Carlos Vullo*, Andrea Rocha, Carola Romanini, Magdalena Romero, Laura Catelli, Martina Rotondo, Micaela Longaray EAAF Forensic Genetic Lab, Independencia, 644-3A, Cordoba, Argentina
A R T I C LE I N FO
A B S T R A C T
Keywords: Bone sampling Macroscopic bone aspect DNA yield STR typing results
The Argentine Forensic Anthropology Team (EAAF) investigates human rights violations which took place in Argentina during the military government that ruled out from 1976 to 1983 and also cooperates in other countries with humanitarian crisis as well. Most of the bone remains that the EAAF analyses show long post mortem interval. Consequently, the skeletal remains may have been exposed to different conditions such as moisture, temperature and pH conditions, microorganisms and different types of soil that can produce various degrees of preservation. On the other hand, severely burnt remains can make the genetic analysis more complex. All the above mentioned influences the DNA quantity and quality. In the present study, we scored the samples to investigate the correlation between the bone macroscopic aspect and the concentration of the DNA extracted from it. Samples were classified into four major groups: score 1 was assigned to samples with very good preservation, score 2 to those showing slight superficial alterations. Score 3 and 4 samples showed regular and bad macroscopic appearance, respectively. Our results clearly supports that samples with good preservation show high well-preserved DNA concentrations and also a higher number of reportable STR markers in comparison to those ranked in the worst categories. This work proposes a useful guide that can help anthropologists to make a more efficient selection of aged-samples for genetic studies.
1. Introduction The main problems of DNA extraction from skeletal material are DNA degradation due to environmental factors and the presence of soilderived inhibitors like humic acids. The quality of DNA preservation in these samples is highly variable and often limited and/or degraded. Bone and teeth clearly protect DNA from environmental degradation and biological attack through their physical and chemical robustness. It seems reasonable to suppose that the characteristics of skeletal remains that are correlated with their long term survival would be those that contribute to the protection of DNA from degradation. Because of this, it is expected that samples with a good state of preservation will lead to high quantity and quality DNA recovery in comparison to those samples with a poor visual aspect. The aim of this study is to evaluate the macroscopic aspect of different types of skeletal remains and to compare it with STR typing results. The efficiency of macroscopic classification was determined on the basis of bone sample DNA concentration obtained by Real-Time PCR assay and the 15 loci from any of the STR amplification kits used in
⁎
this analysis. These data can be a useful guide for sample selection of skeletal remains with a greater chance of obtaining good results based on their macroscopic appearance. 2. Materials and methods A total of 1727 skeletal remains were analyzed, including teeth and 19 different types of bone pieces. Most of the processed samples have over 35 years of post-mortem interval. Based on our experience, the samples were classified into four categories according to the visual macroscopic aspect of skeletal samples usually received by our laboratory. Totally burned samples were not included because the reproducibility of genetic results is extremely difficult. Score 1 corresponds to samples with very good appearance: bones in this category have few spots and / or pigmentations that can be easily removed or they may not even be present at all. They have a strong firmness and abundant cortical portion. Teeth keep their structure intact without the presence of caries and they may also have a slight coloration.
Corresponding author. E-mail address: [email protected] (C. Vullo).
https://doi.org/10.1016/j.fsigss.2019.10.167 Received 9 September 2019; Accepted 16 October 2019 1875-1768/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Carlos Vullo, et al., Forensic Science International: Genetics Supplement Series, https://doi.org/10.1016/j.fsigss.2019.10.167
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
C. Vullo, et al.
Score 2 involves samples with a good state of preservation: teeth may have some superficial caries and / or spots that are completely removed during the cleaning, their structure remains intact. Bones can also show superficial alterations that are totally removed during the cleaning. The cortical portion is still generous. Score 3 applies to regular visual appearance samples: bones show marked alterations in their surface and their structure is compromised. The cortical fraction decreases significantly and the sample adopts a sandy appearance that contributes to a shedding of abundant dust particles during the cleaning and, therefore, a large quantity of material is lost. Teeth samples may present deep caries with highly dark spots; and the roots, in most of the cases, present open ends. During the cleaning process of this type of samples there is no great improvement in their appearance since the mentioned alterations persist. Score 4 includes pieces with a poor state of preservation: bones have a fragile-sandy appearance and even scales can be observed due to the alteration of their structure. The cortical portion is practically absent after cleaning. Teeth can be compromised due to the abundant holes in their structure, numerous spots, fractures and one or more roots broken leaving their interior exposed. Samples in this category are extremely fragile and tend to break while they are being cleaned or at the slightest contact with any other material. Superficial alterations include the presence of fungi, coloration due to the type of soil and/or presence of crystals that may have also been on the ground, as well as perforations due to termites, among others. Fig. 1. Examples of bones and teeth for all the proposed scores (above) and average DNA concentration and reportable STR for each score.
2.1. Sample preparation All samples were mechanically cleaned, washed, exposed to 10% sodium hypochlorite for 10 min and washed again; finally briefly rinsed with 96% ethanol and irradiated with UV-light (254 nm) for 15 min per side. Samples were pulverized using 6770 Freezer Mill (SPEX CertiPrep, Metuchen, NJ, USA) [1].
3. Results and discussion Real-Time PCR quantification revealed that samples with a very good and good visual appearance (scores 1 and 2) have higher DNA concentration values than those with a regular or bad appearance (scores 3 and 4) regardless of the extraction method used. On the other hand, it can be observed that the average number of genetic markers reported is directly related to DNA concentration [7] (Fig. 1). Low DNA concentration obtained for regular and bad appearance samples can be explained by the fact that their structure is not preserved and therefore, DNA is totally exposed to severe environmental conditions. In contrast, skeletal remains with very good and good macroscopic aspect presented a considerably higher DNA concentration as their structure still maintains all the characteristics that protect the DNA from external agents. Therefore, these samples have less degraded DNA than those with opposite characteristics. The highest DNA recovery was observed on bones with dense cortical fraction, intact structure and no several superficial alterations. The same results were found for teeth that had all their roots without open end or major caries and/or spots. A significant correlation (p < 0.05; Tukey-Kramer Multiple Comparison Test) between the described scores was found.
2.2. DNA extraction methods This study extends over several years of work and our laboratory has used two different extraction methods during that period: A) Qiagen Blood Maxi Kit (Qiagen, Valencia, CA, USA) and B) PrepFiler BTA Forensic Extraction Kit (Applied Biosystems, Foster City, CA, USA) on Automate Express platform. In both protocols the amount of starting material was increased. For extraction method A, samples were previously demineralized with 0.5 M EDTA pH 8 at room temperature for 72 h with rotation movements [2,3]. For method B, bone powder was treated with lysis buffer (0.5 M EDTA and 1% N-Laurylsarcosinate), proteinase K 20 mg/mL (Invitrogen, Carlsbad, CA, USA) and 1.0 M DTT (Sigma-Aldrich) and incubated overnight at 56 °C for full demineralization [4–6]. 2.3. DNA quantification and amplification
4. Conclusions Human genomic DNA was determined by using the Quantifiler™ Human DNA Quantification Kit or Quantifiler™ Human Plus DNA Quantification Kit (Applied Biosystems, Foster City, CA, USA) and 7500 Real-Time PCR System (Applied Biosystems). In order to compare the data obtained with both kits, the information of the smaller amplicon of the autosomal target for Quantifiler™ Human plus DNA Quantification Kit was taken into account. PCR amplification was carried out by using AmpFLSTR™ Identifiler™ Plus PCR Amplification Kit, GlobalFiler™ PCR Amplification Kit (Applied Biosystems, Foster City, CA, USA) and PowerPlex® Fusion System (Promega, Madison, WI, USA). Only STR markers shared by these kits were considered in the present study. Amplified products were separated and detected on ABI PRISM 3130 Genetic Analyzer or 3500 Genetic Analyzer (Applied Biosystems).
Our results show a significant correlation between described scores based on the macroscopic appearance of bone samples with DNA recovery and the number of reportable STRs. This work proposes a guide useful for anthropologists to make a more efficient sample selection of aged-samples, contributing to DNA typing successs. References [1] W. Goodwin, Forensic DNA Typing Protocols, second edition, Humana Press, New York, 2016. [2] J. Davoren, D. Vanek, R. Konjhodzić, et al., Highly effective DNA extraction method for nuclear short tandem repeat testing of skeletal remains from mass graves, Croat. Med. J. 48 (4) (2007) 478-85. [3] C. Romanini, M.L. Catelli, A. Borosky, et al., Typing short amplicon binary
2
Forensic Science International: Genetics Supplement Series xxx (xxxx) xxx–xxx
C. Vullo, et al.
London, 2011. [6] O. Loreille, T.M. Diegoli, J.A. Irwin, M.D. Coble, T.J. Parson, High efficiency DNA extraction bone by total demineralization, Forensic Sci. Int. Genet. 1 (2006) 191-5. [7] A. Milos, A. Semanovic, L. Smajlovic, et al., Success rates of nuclear STR typing from different skeletal elements, Croat. Med. J. 48 (2007) 486-93.
polymorphisms: supplementary SNP and Indel genetic information in the analysis of highly degraded skeletal remains, Forensic Sci. Int. 6 (4) (2012) 469–476. [4] S. Amory, R. Huel, A. Bilic´, et al., Automatable full demineralization DNA extraction procedure from degraded skeletal remains, Forensic Sci. Int. Genet. 6 (3) (2012) 398–406. [5] J.M. Butler, Advanced Topics in Forensic DNA Typing: Methodology, Academic,
3