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The generation of a DNA profile from skeletal remains is an important part of the identification process in both mass disaster and unidentified person cases. Since bones and teeth are often the only biological materials remaining after exposure to environmental conditions, intense heat, certain traumatic events and in cases where a significant amount of time has passed since the death of the individual, the ability to purify large quantities of informative DNA from these hard tissues would be beneficial. Since sampling the hard tissues for genetic analysis is a destructive process, it is important to understand those environmental and intrinsic factors that contribute to DNA preservation. This will serve as a brief introduction to these topics, since skeletal sampling strategies and molecular taphonomy have been discussed in depth elsewhere. Additionally advances in skeletal DNA extraction and analysis will be discussed. Currently there is great variation in the DNA isolation methods used by laboratories to purify DNA from the hard tissues; however, a standardized set of short tandem repeat (STR) loci is analyzed by many US laboratories to allow for comparisons across samples and jurisdictions. Recent advances have allowed for the generation of DNA profiles from smaller quantities of template DNA and have expanded the number of loci analyzed for greater discriminatory power and predictions regarding the geographic ancestry and phenotype of the individual. Finally, utilizing databases and expanding the number of comparison samples will be discussed in light of their role in the identification process.

DNA analysis plays a crucial role in forensic investigations, helping in criminal cases, missing persons inquiries, and archaeological research. This study focuses on the DNA concentration in different skeletal elements to improve human identification efforts. Ten cases of unidentified skeletal remains brought to the Institute of Forensic Medicine in Timisoara, Romania, underwent DNA analysis between 2019 and 2023. The results showed that teeth are the best source for DNA extraction as they contain the highest concentration of genetic material, at 3.68 ng/µL, compared to the petrous temporal bone (0.936 ng/µL) and femur bone (0.633 ng/µL). These findings highlight the significance of teeth in forensic contexts due to their abundant genetic material. Combining anthropological examination with DNA analysis enhances the understanding and precision of identifying human skeletal remains, thus advancing forensic science. Selecting specific skeletal elements, such as the cochlea or teeth, emerges as crucial for reliable genetic analyses, emphasizing the importance of careful consideration in forensic identification procedures. Our study concludes that automated DNA extraction protocols without liquid nitrogen represent a significant advancement in DNA extraction technology, providing a faster, more efficient, and less labor-intensive method for extracting high-quality DNA from damaged bone and tooth samples.

Background/Objectives: DNA profiling can fail, or produce poor results, when naturally occurring materials are present during the amplification step. This study demonstrates that simple modifications to the reaction setup can overcome this obstacle. PCR inhibition is caused by a range of compounds including haem, humic acid and dyes. Various strategies to overcome this inhibitory effect have been explored, such as improving extraction methods to remove these compounds, diluting samples to reduce inhibitor concentration, or using inhibitor-tolerant DNA polymerases. In this study, we evaluate whether modified setups can help mitigate the effects of humic acid, a common inhibitor that induces various inhibition mechanisms. Methods: We combined the GlobalFiler STR kit with Investigator Quantiplex Pro into a single reaction. Supplementing the amplification GlobalFiler with additional reagents creates altered amplification environments that use additional DNA polymerase and reaction buffer. Results: The modified setups outperformed the standard GlobalFiler protocol, even at the highest concentration of humic acid tested. The STR reactions supplemented with qPCR reagents produced higher-quality profiles with improved allele amplification and an even peak balance, indicating that a dual-DNA polymerase system offers a more robust and inhibitor-tolerant environment for STR amplification. In addition to demonstrating the value of this combined approach, these data provide a comprehensive dataset characterising the impact of increasing humic acid concentrations on profile quality from an ideal DNA input. Conclusions: For PCR inhibitors with similar mechanisms this approach offers broader applicability in forensic casework and a promising step toward more reliable and robust profiling of inhibited samples.

The interpretation and statistical evaluation of mixed DNA profiles often presents a particular challenge in forensic DNA investigations. Only in specific combinations can single cellular components of a mixture be assigned to one contributor. In this study, the DEPArray™ technology, which enables image-assisted immunofluorescent-sorting of rare single cells using dielectrophoretic (DEP) forces, was applied together with different preliminary tests to identify the individual/s who contributed blood to a given mixture. The technique was successfully applied in two routine casework samples. In order to ascertain how old a stain can be and still be processed successfully, white blood cells from two 10- and one 27-year-old stains were investigated. Depending on the stain’s age, the associated DNA degradation level and the number of target cells successfully isolated, the final profile reflects a compromise between the gain of information due to isolation of pure cells of a specific cell type from a single contributor and the loss of discriminatory power due to incomplete profiles caused by DNA degradation.

DNA is now routinely used in criminal investigations and court cases, although DNA samples taken at crime scenes are of varying quality and therefore present challenging problems for their interpretation. We present a statistical model for the quantitative peak information obtained from an electropherogram of a forensic DNA sample and illustrate its potential use for the analysis of criminal cases. In contrast with most previously used methods, we directly model the peak height information and incorporate important artefacts that are associated with the production of the electropherogram. Our model has a number of unknown parameters, and we show that these can be estimated by the method of maximum likelihood in the presence of multiple unknown individuals contributing to the sample, and their approximate standard errors calculated; the computations exploit a Bayesian network representation of the model. A case example from a UK trial, as reported in the literature, is used to illustrate the efficacy and use of the model, both in finding likelihood ratios to quantify the strength of evidence, and in the deconvolution of mixtures for finding likely profiles of the individuals contributing to the sample. Our model is readily extended to simultaneous analysis of more than one mixture as illustrated in a case example. We show that the combination of evidence from several samples may give an evidential strength which is close to that of a single-source trace and thus modelling of peak height information provides a potentially very efficient mixture analysis.

In forensic crime scene investigations, biological fluids such as blood are commonly found in soil. However, the analysis of blood-stained soil can be challenging due to the presence of inhibitors which limit the effective extraction and amplification of the deoxyribonucleic acid (DNA) required to produce a reportable DNA profile. There are some extraction methods that have been applied to blood-stained soil in forensic science, but these have produced sporadic results. This research has taken a number of different extraction methods from the fields of ancient DNA and environmental DNA and broken them down into the individual steps of pre-treatment, incubation, separation and purification. These steps were assessed independently then combined into various extraction methods to determine the best technique that can effectively and reliably profile human DNA from blood-stained soil. Testing involved assessment of three extraction buffers, (cetyltrimethylammonium bromide, guanidine thiocyanate, and proteinase K), four pre-treatment methods, (polyvinylpyrrolidone, ethylenediaminetetraacetic acid, hydrochloric acid, and sodium hydroxide), three separation steps, (centrifugation, phenol chloroform, and chloroform) and four purification steps, (size exclusion chromatography, bind elute columns, isopropanol precipitation and silica magnetic beads). The most effective procedure was found to be a polyvinylpyrrolidone pre-treatment with a proteinase K extraction buffer followed by magnetic silica bead purification with or without centrifugation. However, centrifugation separation was found to be equally effective after the pre-treatment step as after the incubation step. Our results shows that most of the current forensic procedures would benefit from the addition of a pre-treatment step prior to processing through the automated DNA profiling pipeline. •A proteinase K with silica bead purification is best for the extraction and profiling of human DNA from blood-stained soil.•A PVP pre-treatment prior to DNA extraction improves the ability to generate a human DNA profile from blood-stained soil.•A PVP pre-treatment step can be added to any current proteinase K extraction method currently being used in forensic science.

Forensic DNA profiles are established by multiplex PCR amplification of a set of highly variable short tandem repeat (STR) loci followed by capillary electrophoresis (CE) as a means to assign alleles to PCR products of differential length. Recently, CE analysis of STR amplicons has been supplemented by high-throughput next generation sequencing (NGS) techniques that are able to detect isoalleles bearing sequence polymorphisms and allow for an improved analysis of degraded DNA. Several such assays have been commercialised and validated for forensic applications. However, these systems are cost-effective only when applied to high numbers of samples. We report here an alternative, cost-efficient shallow-sequence output NGS assay called maSTR assay that, in conjunction with a dedicated bioinformatics pipeline called SNiPSTR, can be implemented with standard NGS instrumentation. In a back-to-back comparison with a CE-based, commercial forensic STR kit, we find that for samples with low DNA content, with mixed DNA from different individuals, or containing PCR inhibitors, the maSTR assay performs equally well, and with degraded DNA is superior to CE-based analysis. Thus, the maSTR assay is a simple, robust and cost-efficient NGS-based STR typing method applicable for human identification in forensic and biomedical contexts.

Who is a Native American? And who gets to decide? From genealogists searching online for their ancestors to fortune hunters hoping for a slice of casino profits from wealthy tribes, the answers to these seemingly straightforward questions have profound ramifications. The rise of DNA testing has further complicated the issues and raised the stakes. In Native American DNA, Kim TallBear shows how DNA testing is a powerful-and problematic-scientific process that is useful in determining close biological relatives. But tribal membership is a legal category that has developed in dependence on certain social understandings and historical contexts, a set of concepts that entangles genetic information in a web of family relations, reservation histories, tribal rules, and government regulations. At a larger level, TallBear asserts, the \"markers\" that are identified and applied to specific groups such as Native American tribes bear the imprints of the cultural, racial, ethnic, national, and even tribal misinterpretations of the humans who study them. TallBear notes that ideas about racial science, which informed white definitions of tribes in the nineteenth century, are unfortunately being revived in twenty-first-century laboratories. Because today's science seems so compelling, increasing numbers of Native Americans have begun to believe their own metaphors: \"in our blood\" is giving way to \"in our DNA.\" This rhetorical drift, she argues, has significant consequences, and ultimately she shows how Native American claims to land, resources, and sovereignty that have taken generations to ratify may be seriously-and permanently-undermined.

Evaluations of forensic observations considering activity level propositions are becoming more common place in forensic institutions. A measure that can be taken to interrogate the evaluation for robustness is called sensitivity analysis. A sensitivity analysis explores the sensitivity of the evaluation to the data used when assigning probabilities, or to the level of uncertainty surrounding a probability assignment, or to the choice of various assumptions within the model. There have been a number of publications that describe sensitivity analysis in technical terms, and demonstrate their use, but limited literature on how that theory can be applied in practice. In this work we provide some simplified examples of how sensitivity analyses can be carried out, when they are likely to show that the evaluation is sensitive to underlying data, knowledge or assumptions, how to interpret the results of sensitivity analysis, and how the outcome can be reported. We also provide access to an application to conduct sensitivity analysis. •Forms of sensitivity analysis that apply to activity level evaluations are presented.•Examples are given for when evaluations are likely to be most sensitive to data.•Strategies for practically dealing with sensitivity evaluations are discussed.•Examples of reporting the results of sensitivity evaluations are provided.

Y-chromosomal short tandem repeat (Y-STR) markers are routinely used in forensic casework to identify male donors of biological traces left at crime scenes, particularly in sexual assault cases. However, the evidential value of a match between the Y-STR profile of a trace and a potential donor, usually a crime suspect, is difficult to quantify, and the common albeit inappropriate practise to equate Y-STR match probabilities with Y-STR profile frequencies estimated from population databases has been subject to scientific debate for decades. As a solution to this long-standing problem, we suggest an alternative approach to the calculation of Y-STR match probabilities that involves splitting the group of potential donors other than the suspect into two: (i) his close male relatives (termed his ‘pedigree’) and (ii) all other males. While an upper limit to the match probability is easily calculated for the second group, it is computationally challenging to derive for the first. We therefore developed a mathematical framework that uses importance sampling to reconstruct and evaluate the Y-STR profiles of untyped members of the suspect’s pedigree by way of simulation. Extensive testing with elementary pedigrees of different structure and complexity confirmed that both, the framework and its Python-based software implementation yield match probability estimates that approximate well the correct analytical results, depending upon the number of simulations performed. Our methodology thus facilitates a more appropriate and valid solution to the long-standing problem of interpreting Y-STR profile matches in forensic casework.