Determination of DNA yield rates in six different skeletal elements in ancient bones

Current sampling strategy for laboratories typing bones for human identification include samples obtained from femur, tooth and temporal bone. Latest studies suggest that the small bones of the hands and feet were very similar or even better in DNA yield. These bones can be easily sampled with a disposable scalpel and thus reduce potential DNA contamination. The aim of our study was to determine the suitability of metatarsals, metacarpals and phalanges for genetic identification. 48 bone samples from 8 different skeletons (six from 18^th century and two from 3rd century) were obtained from 5 archaeological sites in Slovenia. In each skeleton, 6 different skeletal elements were sampled (temporal bone, molar, femur, metacarpal bone, metatarsal bone and proximal phalanx of the hand), and strict precautions followed to prevent contamination. Half of gram of bone powder was decalcified using full demineralization extraction method. The DNA was purified in a Biorobot EZ1 (Qiagen), DNA content determined with the PowerQuant kit (Promega), and autosomal STR typing performed with the Investigator ESSplex Plus kit (Qiagen). Up to 8.75 ng DNA/g of powder was obtained from samples analyzed. The highest yields were detected in temporal bone and the lowest in femur. The success rate of STR typing was evaluated according to the number of successfully typed loci and a strong correlation between the success rate of STR typing and the amount of extracted DNA was confirmed. For all eight skeletons full consensus genetic profiles were determined from skeletal elements analyzed. Our findings suggest it would be suitable to include metatarsal and metacarpal bones in sampling strategy for human identification although further research is needed to substantiate the findings of this study.     

DNA typing from skeletal remains using GlobalFiler™ PCR amplification and Investigator® 24plex QS kits

Since the beginning of our work in 2003 our laboratory has focused exclusively on STR DNA from bone, a powerful tool in missing person cases. In cases such as mass disasters or missing persons, human remains are challenging to identify as they may be fragmented, burnt, recovered from water, degraded, and/or contain inhibitory substances. To address these challenges, this study has evaluated the performance of relatively new STR kits Investigator® 24plex QS kit (Qiagen) and GlobalFiler™ PCR Amplification kit (Thermo Fisher Scientific) by comparing it with current uses of the AmpFLSTR® Identifiler® Plus kit (Applied Biosystems) to obtain genetic information from skeletal remains. We analyzed 20 bone samples of skeletal remains from routine casework submitted for body identifications by law enforcement corresponding using Investigator® 24plex QS kit and GlobalFiler™ PCR Amplification kit, previously analysed AmpFLSTR® Identifiler® Plus kit (Thermo Fisher Scientific). The data indicates that the STR profiles obtained using the GlobalFiler™ and Investigator® 24plex QS kit for analysis of skeletal remains has shown results in an increased number of reportable genetic loci, and provide greater power of discrimination in comparison to the Identifiler® Plus Kit. Advanced extraction and purification techniques, together with more sensitive and robust new amplification kits allowed us to overcome the challenges associated with processing compromised skeletal remains and ultimately obtain full STR DNA profiles in 99% of the bones.     

Comparison of DNA yield after long-term storage of Second World War bone samples

Sample storage is of paramount importance in forensic genetics laboratories since only optimal storage enables successful recovery of DNA from old bones that contain very low amount of severely degraded DNA. When identification of missing persons from skeletal remains is completed, bone sample is routinely stored at -20 °C for long-term storage for retesting in future, if necessary. After molecular genetic analyses of Slovenian Second World War (WWII) victims, small fragments of femurs were stored at -20 °C. Reduction in DNA recovery has been observed in frozen liquid DNA extracts by some authors and the goal of our study was to explore how freezing of bone samples affects the preservation of DNA. To achieve this goal, the difference in DNA yield in extracts obtained from WWII bones analyzed in 2009 (data from published paper) and DNA yield in extracts obtained from the same bones (piece sampled next to the one used in 2009) taken out of the freezer after long-term storage on -20 °C for 10 years was examined, using the same extraction method and the same quantification kit. Up to 100 ng DNA/g of bone powder was obtained from 57 WWII femurs and up to 31 ng DNA/g of bone powder from the same femurs investigated after long-term storage in this study. 0,5 g of bone powder was decalcified using full demineralization extraction method. The DNA was purified in a Biorobot EZ1 device (Qiagen) and DNA quantity determined with the Human Quantifiler kit (TFS). Statistical analysis showed significant difference in DNA yield in extracts obtained from WWII bones in 2009 and extracts obtained from the same bones stored at -20 °C after 10 years. As reported for frozen liquid DNA extracts, reduction in recovery of DNA was confirmed for frozen bone samples as well.     

STR analysis of skeletal remains exhumed from a dry “water well” used as a massive clandestine burial in Argentina

The military dictatorship that took power in Argentina from 1976 to 1983, brought enforced disappearance and death to thousands of people in the country. “Pozo de Vargas” is a 3.5 m wide and 60 m deep dry water well used as a massive clandestine burial site in Tucumán, Argentina. The site contained commingled remains as well as partially articulated body parts. Since 2011, EAAF Forensic Genetics Laboratory (LGF-EAAF) has been processing post mortem (PM) samples exhumed from the well to achieve the intra-skeletal re-association and the identification of the bodies or body parts. 981 skeletal remains, with different states of preservation, were analyzed for STR typing using commercial available kits. Different bone elements were exhumed for intra-skeletal re-associations (PM-PM genetic comparisons): teeth, femur and tibia represented more than 65% of sampling and almost all of them were successfully re-associated; spongy bones as scapula, vertebrae and clavicles yielded worse results, although almost 80% of them were also re-associated. 871 skeletal remains (89%) were successfully typed for autosomal STR markers and re-associated into 147 unique STR profiles meaning that at least 147 different individuals were thrown into the water well. After re-association, unique STR profiles obtained from the skeletal remains were compared to ante mortem (AM) family reference database in order to identify victims (AM-PM genetic comparisons), being possible to restore 113 victims to their families. Another challenge of this identification project was the presence of biologically related victims inside the well.     

Highly efficient and automated extraction of DNA from human remains using a modified EZ1 protocol

Bones and teeth often represent the only sources of DNA available for identifying human remains. DNA in bones and teeth is generally better preserved than that in soft tissues because of the presence of hard connective tissue with a high level of calcium. Because of the extensive mineralisation, the choice of an efficient DNA extraction procedure is important to minimise the sampling of a high level of minerals and to remove polymerase chain reaction (PCR) inhibitors. Some protocols are available for DNA extraction from bones and teeth as part of the Qiagen EZ1 DNA Investigator Kit using the EZ1 Advanced XL automated purification platform. To improve the efficiency of DNA extraction from skeletal remains, the present study focuses on a modification to these already available protocols. In this study, different bones and teeth collected between 1 and 50 years after death were subjected to DNA extraction using the standard EZ1 protocol, a supplementary protocol, and a modified protocol. The modified approach included a decalcification step, whereas the Qiagen protocols worked directly on non-decalcified powder. In all three procedures, 150 mg samples were used for DNA extraction. We evaluated the quantity of DNA recovered from samples, the presence of any PCR inhibitors co-extracted, the level of DNA degradation, the quality of short tandem repeat (STR) profiles, and the reproducibility of the modified procedure. When compared with the other protocols, the modified protocol resulted in the best recovery of DNA that was free of PCR inhibitors. Additionally, the STR profiles were reliable and of high quality. In our opinion, the decalcification step increases DNA recovery by softening tissues, which allows lysis solutions to act more effectively. Furthermore, the use of two lysis solutions and the variation added to the EZ1 purification step allow for DNA recovery with quality and quantity superior to those of the previously available Qiagen-based protocols. These findings may be helpful solutions to the problems commonly encountered when dealing with difficult samples, such as bones and teeth.

Key points

• Bones and teeth often represent the only sources of DNA for identifying human remains.
• The choice of an efficient DNA extraction procedure is important for maximizing DNA recovery and removing PCR inhibitors.
• This study focuses on modifications to the previously available Qiagen-based protocols.
• The modified protocol enabled the best recovery of DNA, and both quality and quantity were superior to those of the previously available Qiagen-based protocols.
• The STR profiles obtained from samples extracted using the modified protocol were reliable and of high quality.

     

2种陈旧骨骼、牙齿DNA纯化方法的比较

目的比较有机法+QIAquick纯化法和DNA IQ磁珠法对陈旧骨骼和牙齿DNA的纯化效果。方法选择10份陈旧骨骼和12份牙齿样本,进行消化后分别采用有机法+QIAquick纯化法和DNA IQ磁珠法进行提取纯化,进行DNA定量后用SinofilerTM试剂盒进行检测。结果 2种方法纯化的骨骼、牙齿DNA的IPC CT值无显著差异。有机法+QIAquick纯化法纯化的骨骼、牙齿DNA平均浓度分别为0.180ng/μL±0.068ng/μL和0.132ng/μL±0.027ng/μL,所有样品均获得全部基因分型。DNA IQ磁珠法纯化的DNA平均浓度分别为0.038ng/μL±0.028ng/μL和0.036ng/μL±0.007ng/μL,有5份骨骼和6份牙齿样本仅获得部分基因分型或未能分型。结论有机法+QIAquick纯化法对陈旧骨骼、牙齿DNA的纯化效果优于DNA IQ磁珠法。     

A Simple and Efficient Method of Extracting DNA from Aged Bones and Teeth

DNA is often difficult to extract from old bones and teeth due to low levels of DNA and high levels of degradation. This study established a simple yet efficient method for extracting DNA from 20 aged bones and teeth (approximately 60 years old). Based on the concentration and STR typing results, the new method of DNA extraction (OM) developed in this study was compared with the PrepFiler? BTA Forensic DNA Extraction Kit (BM). The total amount of DNA extracted using the OM method was not significantly different from that extracted using the commercial kit (p > 0.05). However, the number of STR loci detected was significantly higher in the samples processed using the OM method than using the BM method (p < 0.05). This study aimed to establish a DNA extraction method for aged bones and teeth to improve the detection rate of STR typing and reduce costs compared to the BM technique.     

人骨骼及牙齿DNA提取方法的比较和优化

目的人骨骼和牙齿DNA提取方法的比较和优化。方法收集18份不同个体的长骨、30颗磨牙和同一个体2根股骨、8颗磨牙。利用TissueLyser-Ⅱ组织破碎仪和PreFiler Express BTA^TM法医DNA提取试剂盒（BTA法）,应用Automate Express^TM自动化法医DNA提取系统提取DNA,进行STR分型,与脱钙法进行比较,并进行实验条件优化。结果用TissueLyser-Ⅱ结合BTA法,约2.5h即可完成骨骼和牙齿的DNA提取,分型成功率分别为94.4%和96.7%。与脱钙法比较,两种方法获得DNA质量浓度和检出率比较接近（P〈0.05）,但BTA法在操作过程方面更具优势。最佳样本量为100mg,消化时间为2h。结论采用TissueLyser-Ⅱ组织破碎仪结合BTA法对骨骼和牙齿进行DNA提取和分型检验,能满足实际检案的要求,可在法医学实践中选择使用。     

The Correlation Between Skeletal Weathering and DNA Quality and Quantity*

Abstract: Mitochondrial DNA analysis of skeletal material is invaluable in forensic identification, although results can vary widely among remains. Previous studies have included bones of different ages, burial conditions, and even species. In the research presented, a collection of human remains that lacked major confounders such as burial age, interment style, and gross environmental conditions, while displaying a very broad range of skeletal degradation, were examined for both mitochondrial DNA (mtDNA) quality and quantity. Overall skeletal weathering, individual bone weathering, and bone variety were considered. Neither skeletal nor bone weathering influenced DNA quality or quantity, indicating that factors that degrade bone do not have the same effect on DNA. In contrast, bone variety, regardless of weathering level, was a significant element in DNA amplification success. Taken together, the results indicate that neither skeletal nor individual bone appearance are reliable indicators of subsequent mtDNA typing outcomes, while the type of bone assayed is.     

Full STR profile of a 67-year-old bone found in a fresh water lake

DNA extraction from and DNA typing of fresh water-exposed aged bone specimens poses a challenging task and is not very well examined. This study presents a new method to extract typable DNA from such problematic bone specimens. The procedure comprises low-heat drilling and cryogrinding, mild lysis conditions, and silica-column-based DNA cleaning. DNA quantity is assessed by quantitative PCR prior to short tandem repeat (STR) amplification. The procedure was employed with a 67-year-old tibia bone fragment recovered from a fresh water lake and succeeded to produce a full STR profile using the MPX-SP1 and MPX-SP2 mini-STR kits and a partial profile with 12 successfully amplified STRs using the Identifiler STR kit. The new method for the extraction of DNA from aged fresh water-exposed bone specimens presented herein was successfully applied to prepare DNA of sufficient quality and quantity to generate a full STR profile.     

An analysis of data curated from 5 years of identifying human remains

An archive of 5 years of cases involving the identification of human remains was curated, collecting information on: The sample type submitted, the number of STR loci yielding interpretable results, the kinship challenge posed, and the outcome for the case. A total of 129 cases of remains ID were investigated using manual DNA extraction and recovery methods with amplification of STR markers using the Power Plex 21 multiplex STR kit from Promega Corp. In 52 cases, blood spots collected by the ME were provided as sample and in 100% of those cases, probabilities of relatedness to the reference samples was ≥99%. In 77 cases, tissue other than blood was provided as a source of DNA. These other samples were grouped categorically into long bones (femur and tibia; 40 cases), skull bones/teeth (11 cases), other bones (16 cases), and tissue (normally adherent to bone) (10 cases). Reference samples provided for cases included alleged parents or child(ren) of the victim (86 cases), alleged full siblings of the victim (38 cases), or alleged second-order relatives (five cases). The overall success rate in confirming the identity of the source of the remains in these cases was 89.2%. Our results demonstrate that a laboratory can be often successful identifying human remains using methods easily implemented in any DNA typing laboratory.     

两种骨骼及牙齿纯化方法的比较分析

目的比较硅珠法和硅胶膜法对骨骼和牙齿的纯化效果。方法选择6根骨骼和8颗牙齿,进行消化后分别采用硅珠法与硅胶膜法进行纯化,用Global Filer~(?)试剂盒进行扩增检测,通过比较检出率和峰高来评价这两种方法。结果两种纯化方法均成功检测出了骨骼和牙齿的STR分型。同一样本中,相同基因座上两种方法检出的等位基因分型结果完全一致。两种方法处理得到的平均峰高差异无统计学意义。结论硅胶膜法在骨骼及牙齿的纯化中能满足实际常规检案的要求,且和硅珠法无明显差异,但在操作上更具优势,缺点是成本较高,在实际工作中可以选择使用。     

DNA Profiling Success Rates from Degraded Skeletal Remains in Guatemala

No data are available regarding the success of DNA Short Tandem Repeat (STR) profiling from degraded skeletal remains in Guatemala. Therefore, DNA profiling success rates relating to 2595 skeletons from eleven cases at the Forensic Anthropology Foundation of Guatemala (FAFG) are presented. The typical postmortem interval was 30 years. DNA was extracted from bone powder and amplified using Identifiler and Minifler. DNA profiling success rates differed between cases, ranging from 50.8% to 7.0%, the overall success rate for samples was 36.3%. The best DNA profiling success rates were obtained from femur (36.2%) and tooth (33.7%) samples. DNA profiles were significantly better from lower body bones than upper body bones (p = <0.0001). Bone samples from males gave significantly better profiles than samples from females (p = <0.0001). These results are believed to be related to bone density. The findings are important for designing forensic DNA sampling strategies in future victim recovery investigations.     

DNA typing of human remains found in damp environments

DNA typing is often used to determine identity from human remains. The environment to which the material has been exposed, however, is crucial for the success of the investigation. Damp conditions in particular can cause a rapid degradation of DNA, even in bone and teeth, and thus reduce the chances of successful typing. Here, we present the results of investigations performed on four skulls that had been lying in a damp environment for periods ranging from almost 1 year to about 45 years. In none of these cases was DNA typing successful on bone or, where present, on teeth. Where remnants of brain tissue were used, however, complete STR typing was possible in one case, partial STR typing in another, and mtDNA sequencing could be carried out in three cases. These findings suggest that brain tissue is relatively resistant to putrefaction in damp environments and, unlike bone, appears to exhibit a certain degree of protection against DNA degradation.     

Storage of Second World War bone samples: Bone fragments versus bone powder

Bone samples may yield low-quality and low-quantity DNA and duplicated analyses of different genetic markers have to be performed for identification of missing persons. Mostly no DNA extract is left after analyses and efficient storage of bones is needed to ensure the stability of the sample over time for retesting using new markers and new technologies. Usually not all of the bone powder prepared in grinder is used for extraction and rest can be stored for future analyses. After molecular genetic analyses of 88 victims of Second World War (WWII) Konfin I mass grave in Slovenia (performed in 2009), fragments of femurs and bone powder that were left were stored at -20 °C. Some authors reported that long-term storage of powder results in the reduction of DNA preservation and its degradation (even at low temperature), explained by an increase in oxidative damage as a result of the enormous increase in exposed surface area. Consequently, grinding of bones as shortly prior to DNA extraction was recommended. The goal of our study was to explore the difference in DNA yield between bone fragment and bone powder frozen for 10 years. 57 WWII femurs were examined and DNA extracted from each of them using bone fragment (piece sampled next to the one used in 2009) and bone powder obtained in 2009, both taken out of freezer after 10 years of storage. Half gram of bone powder was decalcified using full demineralization extraction method. The DNA was purified in a Biorobot EZ1 (Qiagen) and quantified with PowerQuant kit (Promega). Statistical analysis showed significant difference at the 0.05 level in DNA yield comparing fragments of bones and bone powder stored at -20 °C for 10 years. The results show there is more DNA stored in the bone powder than in the bone fragments. Because of time - consuming powdering procedure we recommend to store not only the fragment of the bone, but obtained bone powder as well.     

Preparation of degraded human DNA under controlled conditions

DNA typing through analysis of short tandem repeats (STRs) and mitochondrial DNA (mtDNA) by means of the polymerase chain reaction (PCR) and sequencing are the common methods for the forensic identification of persons and reconstruction of kinship, especially when skeletal human remains have to be analyzed. Furthermore, samples typically found at crime scenes may be both quantitatively and qualitatively inadequate since they may contain very scarce and often degraded DNA due to exposure to heat, light, humidity, and microorganisms. In order to improve the performance of STR typing technology in those cases where DNA availability is limited, it would be desirable to have a source of degraded DNA with known properties. For this purpose, we have developed a method to prepare artificially degraded DNA under controlled conditions. By treatment of genomic DNA with sonication and DNAse I we have produced DNA fragments within a defined range of lengths. STR typing of this degraded DNA with a commercially available multiplex kit could only produce partial profiles as indicated by the absence of STR alleles with sizes >200 bp. This artificially degraded DNA can be used for the improvement and standardization of STR typing protocols when only highly degraded DNA is available for analysis.     

Teeth as a source of DNA for forensic identification of human remains: A Review

Teeth and bones are frequently the only sources of DNA available for identification of degraded or fragmented human remains. The unique composition of teeth and their location in the jawbone provide additional protection to DNA compared to bones making them a preferred source of DNA in many cases. Despite this, post-mortem changes in the structure and composition of teeth, and the location and diagenesis of DNA within them are poorly understood. This review summarises current knowledge of tooth morphology with respect to DNA content and preservation, and discusses the way in which post-mortem changes will affect the recovery of DNA from teeth under a range of commonly used extraction protocols. We highlight the benefits and pitfalls of using specific tooth tissues for DNA extraction and make recommendations for tooth selection and sampling that will maximise DNA typing success. A comprehensive understanding of tooth structure and an appreciation of the relationship between DNA and mineralized tissues in post-mortem teeth are critical for optimal sample selection. More informed sampling methods that target specific tooth tissues will increase the likelihood of successful genetic analysis and allow for efficient and timely missing persons case work and disaster victim identification response.     

自动荧光分析法检验陈旧骨骼和牙齿的vWA和LPL位点

为鉴定陈旧骨骼和牙齿的尸源 ,对DNA的提取方法进行了研究,并建立了vWA和LPL位点的自动荧光分析技术,该法简便、快速、有效、可对存放2、3、4、7年的陈旧骨骼和牙齿成功地进行检验,在检验中能直接确定样品的等位基因     

Nuclear DNA typing from ancient teeth

Because of the adverse effects that diagenesis exert on ancient skeletal remains, DNA from these samples is often compromised to the point where genetic typing can be challenging. Nevertheless, robust and reliable methods are currently available to allow successful genotyping of ancient specimens. Here we report nuclear DNA-based methods and typing strategies used to analyze 2 human skeletons from a medieval burial. Reliable DNA nuclear profiles were obtained from teeth, whereas mitochondrial DNA analyses in bones were inconclusive. A complete nuclear mini short tandem repeat profile was obtained from a well-preserved premolar, but only a partial one from the femur. Increasing the sensitivity of the polymerase chain reaction system allowed a full profile from the latter, but the presence of artifacts reinforced the idea that the interpretation of this kind of analysis must be performed with caution. The results presented here also indicate that DNA from dental pieces can be better preserved than from bones, even in the case of well-preserved long bones with thick cortical tissue such as the femurs, and have a better chance of successful genetic typing, probably because of the high degree of protection conferred to the DNA by the enamel.     

Next generation sequencing technology in Second World War victim identification

The killings during the Second World War (WWII), with nearly 100,000 victims, is one of the greatest losses of life in Slovenia’s modern history and most of the victims are still buried in hidden mass graves and remain unidentified. Identity, ancestry, and phenotypic SNPs, as well as STR markers are already used for solving various cases with Next Generation Sequencing (NGS) technology. In this study, the Precision ID GlobalFiler NGS STR panel was used to identify the WWII victim that could not be identified with capillary electrophoresis (CE) analyses because limited statistical support was obtained after amplification of autosomal STRs using CE STR kits. Bones and teeth were analysed and compared to family references (nephew and niece on paternal line). Prior to DNA isolation 0.5 g of powder was decalcified. The DNA was purified in a Biorobot EZ1 device. The nuclear DNA of the samples was quantified with the PowerQuant kit. Because the recommended posterior probability (PP) of 99.9% was followed with the goal of high confidence of correct identification, the NGS STR Panel was used, and after the analysis of additional STR loci the statistical calculation showed a PP of 99.99986%, showing that a large enough number of genetic markers were analysed when identifying the skeletal remains of the aunt. PP value endorsed the hypothesis that the tooth and bone samples were from individual related to the family references rather than from unrelated individual. In presented case, NGS technology proved to be a powerful tool for increasing the number of autosomal STRs needed for identification of WWII victims when linear markers cannot be used for comparison and only distant relatives are available for analyses.