Characterization of sequence variations in the extended flanking regions using massively parallel sequencing in 21 A-STRs and 21 Y-STRs.

In forensic genetics, utilizing massively parallel sequencing (MPS) to analyze short tandem repeats (STRs) has demonstrated several advantages compared to conventional capillary electrophoresis (CE). Due to the current technical limitations, although flanking region polymorphisms had been mentioned in several previous studies, most studies focused on the core repeat regions of STRs or the variations in the adjacent flanking regions. In this study, we developed an MPS system consisting of two sets of multiplex PCR systems to detect not only the STR core repeat regions but also to observe variants located at relatively distant positions in the flanking regions. The system contained 42 commonly used forensic STRs, including 21 autosomal STRs (A-STRs) and 21 Y-chromosomal STRs (Y-STRs), and a total of 350 male individuals from a Chinese Han population were genotyped. The length and sequence variants per locus were tallied and categorized based on length (length-based, LB), sequence without flanking region (core repeat regions sequence-based, RSB), and sequence with flanking region (core repeat and flanking regions sequence-based, FSB), respectively. Allele frequencies, Y-haplotype frequencies, and forensic parameters were calculated based on LB, RSB, and FSB, respectively, to evaluate the improvement in discrimination power, heterozygosity, and effectiveness of forensic systems. The results suggested the sequence variations have more influence on A-STRs and could improve the identification ability of MPS-STR genotyping. Concordance between MPS and CE methods was confirmed by using commercial CE-based STR kits. The impact of flanking region variations on STR genotype analysis and potential factors contributing to discordances were discussed. A total of 58 variations in the flanking regions (53 SNPs/SNVs and 5 InDels) were observed and most variations (48/58) were distributed in A-STRs. In summary, this study delved deeper into the genetic information of forensic commonly used STR and advanced the application of massively parallel sequencing in forensic genetics.

Accounting for site-to-site DNA transfer on a packaged exhibit in an evaluation given activity level propositions.

Considering activity level propositions in the evaluation of forensic biology findings is becoming more common place. There are increasing numbers of publications demonstrating different transfer mechanisms that can occur under a variety of circumstances. Some of these publications have shown the possibility of DNA transfer from site to site on an exhibit, for instance as a result of packaging and transport. If such a possibility exists, and the case circumstances are such that the area on an exhibit where DNA is present or absent is an observation that is an important diagnostic characteristic given the propositions, then site to site transfer should be taken into account during the evaluation of observations. In this work we demonstrate the ways in which site to site transfer can be built into Bayesian networks when carrying out activity level evaluations of forensic biology findings. We explore the effects of considering qualitative vs quantitative categorisation of DNA results. We also show the importance of taking into account multiple individual's DNA being transferred (such as unknown or wearer DNA), even if the main focus of the evaluation is the activity of one individual.

Comprehensive body fluid identification and contributor assignment by combining targeted sequencing of mRNA and coding region SNPs.

Forensic genetic analyses aim to retrieve as much information as possible from biological trace material recovered from crime scenes. While standard short tandem repeat (STR) profiling is essential to individualize biological traces, its significance is diminished in crime scenarios where the presence of a suspect's DNA is acknowledged by all parties. In such cases, forensic (m)RNA analysis can provide crucial contextualizing information on the source level about a trace's composition, i.e., body fluids/tissues, and has therefore emerged as a powerful tool for modern forensic investigations. However, the question which of several suspects contributed a specific component (body fluid) to a mixed trace cannot be answered by RNA analysis using conventional methods. This individualizing information is stored within the sequence of the mRNA transcripts. Massively parallel sequencing (MPS) represents a promising alternative, offering not only higher multiplex capacity, but also the typing of individual coding region SNPs (cSNPs) to enable the assignment of contributors to mixture components, thereby reducing the risk of association fallacies. Herein, we describe the development of an extensive mRNA/cSNP panel for targeted sequencing on the IonTorrent S5 platform. Our panel comprises 30 markers for the detection of six body fluids/tissues (blood, saliva, semen, skin, vaginal and menstrual secretion), along with 70 linkage-controlled cSNPs for contributor assignment. It exhibited high reliable detection sensitivity with RNA inputs down to 0.75 ng and a conservatively calculated probability of identity of 0.03 - 6 % for individual body fluid-specific cSNP profiles. Limitations and areas for future work include RNA-related allele imbalances, inclusion of markers to correctly identify rectal mucosa and the optimization of specific markers. In summary, our new panel is intended to be a major step forward to interpret biological evidence at sub-source and source level based on cSNP attribution of a body fluid component to a suspect and victim, respectively.

Forensic efficiency evaluation of a mtDNA whole genome sequencing system constructed with long fragment amplification strategy on DNA nanoball sequencing platform.

Mitochondrial DNA (mtDNA) is an important genetic marker for degraded biological sample identification, maternal pedigree tracing, and population genetic structure study owing to its characteristics of high copy number, anti-degradable ring structure, and maternal inheritance. Whole mtDNA genome sequencing is an optimal method for the analysis of mtDNA polymorphism and heterogeneity because it allows for the comprehensive use of maternal genetic information. However, because of lacking quantitative evaluations for sequencing data, the scientific interpretation standards for mtDNA sequencing results of the previously used sequencing systems are often different, and false positive or false negative results are prone to occur when faced with the interference of nuclear genomic DNA, or the heterogeneities of mtDNA sequence and structure. In this study, we evaluated a novel mtDNA whole genome sequencing system using long fragment amplification strategy on the DNA nanoball (DNB) sequencing platform. This system demonstrated high sequencing quality and specific mtDNA sequencing efficiencies on positive control DNA and FTA bloodstain samples, as the average Q20 and Q30 values of the corresponding samples were 97.17 % and 91.93 %; 97.37 % and 92.48 %, respectively. The mean mapping percentages for the reference sequences of whole genome DNA (wgDNA), mtDNA, and nuclear genomic DNA (ngDNA) in the corresponding samples were 99.98 %, 99.97 %, 0.03 %, and 99.91 %, 99.40 %, 0.60 %; respectively. The average error calling rates for the bases A, C, G, and T of the whole mtDNA genome were 0.2519 %, 0.2550 %, 0.2906 %; and 0.2392 %, respectively. The efficacy of heteroplasmy identification was assessed using a set of theoretical sites with predetermined rates. These sites were created by combining the samples with known mtDNA haplotypes in certain proportions. The absolute errors between observed and theoretical heteroplasmy values were 89.59 %, 74.68 %, 50.20 %, 12.65 %, 8.31 %, and 4.85 %, while the theoretical heteroplasmy values were 5 %, 10 %, 20 %, 80 %, 90 %, and 95 %, respectively. The absolute error exhibited relative stability when the mtDNA sequencing depth exceeded 500×. Furthermore, the system sequencing efficiency was also confirmed among different kinds of samples, and these samples included natural samples (e.g., peripheral blood samples preserved on FTA cards for 2 and 11 years, and on filter paper for 6 and 9 years), degraded samples, sensitivity samples, samples derived from various bodily fluids, and maternal pedigree samples. In summary, the whole mtDNA genome sequencing system used for forensic identification demonstrated high performance in analyzing mtDNA sequence information, and showed significant prospects for forensic application and maternal genetic research.

Developmental validation of the AGCU YNFS Y Kit: A new 6-dye multiplex system with 44 Y-STRs and 5 Y-InDels for forensic application.

With the widespread use of the Y chromosome in genetics, a lot of commercially available Y chromosome kits were developed, validated, and applied to forensic science practice. The AGCU YNFS Y Kit is a new Y chromosome system containing forty-four preferred Y short tandem repeats (Y-STRs) and five common Y-InDels. In this study, the AGCU YNFS Y system was validated to verify its performance by following the guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM). A series of validation experiments included the following parameters: PCR-based studies, sensitivity studies, species specificity studies, stability studies, mixture studies, precision studies, stutter calculation, mutation and statistical analysis, population study, and case samples and degradation studies. The results suggested that appropriately changing PCR amplification conditions did not affect genotyping; the kit had good sensitivity for trace amounts of DNA (0.0625 ng), mixtures of multiple male individuals (minor: major = 1: 9), and three PCR inhibitors (more than 250 µM hematin, 250 ng/µL humic acid and 50 ng/µL tannic acid). The maximum standard deviation of allele size did not exceed 0.1552 reflecting the high accuracy of the system. By this, 87 DNA-confirmed pairs of father-son pairs were also analyzed for mutations. A total of 18 loci were mutated, with mutation rates ranging from 11.5×10-3 to 34.5×10-3 (95% CI 7.2×10-3-97.5×10-3, DYS627 and DYF404S1). In the population study, the haplotype diversity of 87 unrelated individuals was 0.9997, and discrimination capacity was 0.9885. Degradation studies have demonstrated that UV-C light exposure for up to 120 hours has no effect on male blood and semen-vaginal secretion mixtures. However, complete typing could no longer be obtained after 48 hours of UV exposure in single male saliva and in male saliva and female blood mixed samples. Collectively, the AGCU YNFS Y Kit is sensitive and accurate and can play its application value in forensic science practice.

Forensic height estimation using polygenic score in Korean population.

Height is known to be a classically heritable trait controlled by complex polygenic factors. Numerous height-associated genetic variants across the genome have been identified so far. It is also a representative of externally visible characteristics (EVC) for predicting appearance in forensic science. When biological evidence at a crime scene is deficient in identifying an individual, the examination of forensic DNA phenotyping using some genetic variants could be considered. In this study, we aimed to predict 'height', a representative forensic phenotype, by using a small number of genetic variants when short tandem repeat (STR) analysis is hard with insufficient biological samples. Our results not only replicated previous genetic signals but also indicated an upward trend in polygenic score (PGS) with increasing height in the validation and replication stages for both genders. These results demonstrate that the established SNP sets in this study could be used for height estimation in the Korean population. Specifically, since the PGS model constructed in this study targets only a small number of SNPs, it contributes to enabling forensic DNA phenotyping even at crime scenes with a minimal amount of biological evidence. To the best of our knowledge, this was the first study to evaluate a PGS model for height estimation in the Korean population using GWAS signals. Our study offers insight into the polygenic effect of height in East Asians, incorporating genetic variants from non-Asian populations.

Solving the twin paradox-forensic strategies to identify the identical twins.

Identical twins are also called monozygotic twins which originate from the same zygote that possesses the same genetic make-up. To discriminate between identical monozygotic twins, short tandem repeats has not been found effective, therefore, various techniques, including next-generation sequencing (NGS), are applied. Monozygotic twins can be identified through germ line genomes, through speech using deep learning networks, and through epigenetic analysis. Fingerprint analysis has also been used to distinguish between identical twins, as human beings have unique fingerprints. Two distinct levels of fingerprint are used to distinguish between monozygotic twins based upon the differences in the minutiae points. Examination of the methylation pattern of the genome has an enormous potential to differentiate between identical twins, as the methylation of DNA occurs uniquely to each individual. This article offers an insight into the latest methods and techniques used for the differentiation between the identical twins.

Forensic stability evaluation of selected miRNA and circRNA markers in human bloodstained samples exposed to different environmental conditions.

Recently, RNA markers have been used to identify tissue origins of different kinds of body fluids. Herein, circRNA and miRNA markers were carried out to examine the presence or absence of peripheral blood (PB) in bloodstained samples exposed to different external environmental conditions, which mimicked PB samples left at the crime scenes. PB samples were placed on sterile swabs and then exposed to different high temperatures (37°C, 55°C and 95°C) and ultraviolet light irradiation for 0 d, 0.5 d, 1 d, 3 d, and 7 d, ultra-low and low temperatures (-80°C, -20°C, and 4°C) for 30 d, 180 d and 365 d and different kinds of disinfectants. Total RNA was extracted from bloodstained samples under the above different conditions, and the expressions of target RNAs (including miR16-5p, miR451a, circ0000095, and two reference genes RNU6b and 18 S rRNA) were detected by the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method. Results showed that these selected RNA markers could be successfully measured at all observation points with their unique degradation rates, which exhibited relative stability in degraded bloodstained samples exposed to different environmental conditions. This study provides insights into the applications of these studied miRNA and circRNA markers in forensic science.

Recommendations of the DNA Commission of the International Society for Forensic Genetics (ISFG) on short tandem repeat sequence nomenclature.

The DNA Commission of the International Society for Forensic Genetics (ISFG) has developed a set of nomenclature recommendations for short tandem repeat (STR) sequences. These recommendations follow the 2016 considerations of the DNA Commission of the ISFG, incorporating the knowledge gained through research and population studies in the intervening years. While maintaining a focus on backward compatibility with the CE data that currently populate national DNA databases, this report also looks to the future with the establishment of recommended minimum sequence reporting ranges to facilitate interlaboratory comparisons, automated solutions for sequence-based allele designations, a suite of resources to support bioinformatic development, guidance for characterizing new STR loci, and considerations for incorporating STR sequences and other new markers into investigative databases.

DNA profiling in India: Addressing issues of sample preservation, databasing, marker selection, & statistical approaches.

DNA technology is the gold standard with respect to the identification of individuals from biological evidence. The technology offers the convenience of a universally similar approach and methodology for analysis across the globe. However, the technology has not realised its full potential in India due to the lack of a DNA database and lacunae in sample collection and preservation from the scene of crime and victims (especially those of sexual assault). Further, statistical interpretation of DNA results is non-existent in the majority of cases. Though the latest technologies and developments in the field of DNA analysis are being adopted and implemented,very little has been enacted practically to improve optimise sample collection and preservation. This article discusses current casework scenarios that highlight the pitfalls and ambiguous areas in the field of DNA analysis, especially with respect DNA databases, sampling, andstatistical approaches to genetic data analysis. Possible solutions and mitigation measures are suggested.

Hungarian legislation regarding implementing a forensic DNA elimination database.

The inception of forensic DNA elimination database represents a pivotal advancement in forensic science, aiming to streamline the process of distinguishing between DNA found at crime scenes and that of individuals involved in the investigation process, such as law enforcement personnel and forensic lab staff. In subsequent phases, once familiarity with the database is achieved by its administrators and other stakeholders, and they have accrued sufficient experience, the possibility of expanding the database to encompass first responders-including firefighters, paramedics, emergency medical technicians, and other emergency services personnel-can be contemplated. Key challenges in managing these databases encompass the grounds for collecting samples, ensuring the integrity of both samples and profiles, along with the duration of retention, access to the database, and the protocols to follow when a match is found in the database. This paper outlines the conceptual and detailed legislative framework in Hungary, where the forensic DNA elimination database was introduced in 2022.

Kafka's beautiful eyes: Forensic intelligence utilisation of phenotypic information.

Franz Kafka had beautiful eyes. So striking, that many of the famous author's friends and peers commented on them - but quite variously ('dark', 'brown', 'grey' & 'blue'). Eye colour as perceived by an observer is subjective, being influenced by physiological, environmental, and even sociocultural factors. In a policing context, this does not mean that trait information such as eye colour is not valuable (far from it), but that it must be managed carefully. The Australian Federal Police has recently implemented a forensic DNA phenotyping (FDP, aka. physical trait prediction or PTP) capability, utilising massively parallel sequencing DNA technology to predict an individual's eye colour, biogeographical ancestry and sex from a crime scene sample. This information alone is not itself 'intelligence', but can be used to generate intelligence through holistic analyses undertaken within a transdisciplinary, all-source forensic intelligence (FORINT) framework. FORINT outputs posit abductive propositions typically at the activity/offence level, to provide insight and influence decision making. However, the use of predicted traits requires that they are compared to something; all Australian police databases include fields for physical traits, but no uniform standard is applied across all agencies. Moreover, collection is inconsistent and no automated systems are in place to capture such data systematically. Consider the 'Kafka problem': his peers gave multiply divergent descriptions of his eyes. If a Biology unit had predicted the eye colour of an 'unidentified author' using DNA - how would Kafka be confidently nominated as the contributor? We posit three maxims for law enforcement: (1) To expand the operational utility of forensic science in line with police demands, forensic science should operationalise FDP (e.g. operationally to rank a list of persons of interest, focus lines of enquiry in serious & organised crime, or assist with human remains identification). (2) Such advanced biological techniques are best delivered through an all-source FORINT framework, to maximise opportunities and minimise risk. (3) One cannot pursue techno-scientific advancements in isolation; it is also necessary to influence the operational posture for their implementation. In this paper we explore these issues and provide recommendations relating to (a) police practices, (b) image capture systems, and (c) research opportunities. Phenotypic trait prediction has great potential and can be operationalised effectively through a rigorous FORINT framework. However, there is (continual) work to be done to enhance the operational capabilities that are complementary to - but necessary for - effective forensic science contribution to investigations.

Saliva-derived secondary DNA transfer on fabric: The impact of varying conditions.

This study explored secondary DNA transfer involving saliva, a body fluid often encountered in forensic investigations. Various factors were examined to investigate their potential impact on the transfer of DNA from saliva stains deposited onto common types of fabric (cotton, nylon, and towel). We examined varying types of saliva moisture (wet, dry, and rehydrated) and different types of contact (controlled pressure and active/friction pressure) to quantitatively evaluate how such variables could impact transfer and possible conclusions surrounding saliva-derived DNA deposits. The transfer of DNA was generally least pronounced with more absorbent primary fabrics (cotton and towel materials) while a less absorbent primary fabric (nylon) exhibited a greater propensity for DNA transfer. There were significantly higher amounts of transferred DNA (p < 0.05) observed in wet saliva samples compared to dry and rehydrated saliva samples. Further, the use of active pressure (friction) appeared to result in more DNA transfer overall as compared to controlled pressure contact. Experiments conducted with wet saliva and active pressure (friction) demonstrated the highest likelihood of transfer, with the primary nylon and secondary towel fabric combination demonstrating the greatest average transfer percentage of 94.74 %. The variables explored in this study presented multiple combinations wherein a sufficient amount of DNA (≥ 240 pg total) was transferred to the secondary fabric, making it potentially suitable for STR-PCR amplification in our laboratory. The findings from this study indicate that the type of primary fabric receiving the saliva deposit, the type of saliva moisture, the type of secondary fabric and its moisture type, and the type of contact all have the potential to affect the quantity of DNA transferred and recovered. This study provides empirical data on the ease, and to what extent, DNA from saliva transfers between fabrics and aids DNA activity level evaluations. The significance of this research lies in its contribution to expanding our current understanding of DNA transfer involving saliva within forensic science and criminal investigations.

The advent of forensic DNA databases: It's time to agree on some international governance principles!

National forensic DNA databases are a valuable investigative tool, that have the potential to increase the efficacy of criminal investigations. Their unfettered expansion in recent years raises unsettling ethical issues that require close attention. DNA database expansion threatens the rights to privacy, non-discrimination, and equality, and can undermine public trust in government. This perspective piece relies on data from an international mapping study of Forensic DNA Databases to document the expansion of these databases, highlight the ethical issues they raise, and propose key recommendations for more responsible use of this infrastructure.

Analysis of the privacy-performance tradeoff of reference testing in Forensic Investigative Genetic Genealogy.

During an investigation using Forensic Investigative Genetic Genealogy, which is a novel approach for solving violent crimes and identifying human remains, reference testing-when law enforcement requests a DNA sample from a person in a partially constructed family tree-is sometimes used when an investigation has stalled. Because the people considered for a reference test have not opted in to allow law enforcement to use their DNA profile in this way, reference testing is viewed by many as an invasion of privacy and by some as unethical. We generalize an existing mathematical optimization model of the genealogy process by incorporating the option of reference testing. Using simulated versions of 17 DNA Doe Project cases, we find that reference testing can solve cases more quickly (although many reference tests are required to substantially hasten the investigative process), but only rarely (<1%) solves cases that cannot otherwise be solved. Through a mixture of mathematical and computational analysis, we find that the most desirable people to test are at the bottom of a path descending from an ancestral couple that is most likely to be related to the target. We also characterize the rare cases where reference testing is necessary for solving the case: when there is only one descending path from an ancestral couple, which precludes the possibility of identifying an intersection (e.g., marriage) between two descendants of two different ancestral couples.

Extending the discussion on inconsistency in forensic decisions and results.

The subject of inter- and intra-laboratory inconsistency was recently raised in a commentary by Itiel Dror. We re-visit an inter-laboratory trial, with which some of the authors of this current discussion were associated, to diagnose the causes of any differences in the likelihood ratios (LRs) assigned using probabilistic genotyping software. Some of the variation was due to different decisions that would be made on a case-by-case basis, some due to laboratory policy and would hence differ between laboratories, and the final and smallest part was the run-to-run difference caused by the Monte Carlo aspect of the software used. However, the net variation in LRs was considerable. We believe that most laboratories will self-diagnose the cause of their difference from the majority answer and in some, but not all instances will take corrective action. An inter-laboratory exercise consisting of raw data files for relatively straightforward mixtures, such as two mixtures of three or four persons, would allow laboratories to calibrate their procedures and findings.

Dense single nucleotide polymorphism testing revolutionizes scope and degree of certainty for source attribution in forensic investigations.

The field of forensic DNA analysis has experienced significant advancements over the years, such as the advent of DNA fingerprinting, the introduction of the polymerase chain reaction for increased sensitivity, the shift to a primary genetic marker system based on short tandem repeats, and implementation of national DNA databases. Now, the forensics field is poised for another revolution with the advent of dense single nucleotide polymorphisms (SNPs) testing. SNP testing holds the potential to significantly enhance source attribution in forensic cases, particularly those involving low-quantity or low-quality samples. When coupled with genetic genealogy and kinship analysis, it can resolve countless active cases as well as cold cases and cases of unidentified human remains, which are hindered by the limitations of existing forensic capabilities that fail to generate viable investigative leads with DNA. The field of forensic genetic genealogy combined with genome-wide sequencing can associate relatives as distant as the seventh-degree and beyond. By leveraging volunteer-populated databases to locate near and distant relatives, genetic genealogy can effectively narrow the candidates linked to crime scene evidence or aid in determining the identity of human remains. With decreasing DNA sequencing costs and improving sensitivity of detection, forensic genetic genealogy is expanding its capabilities to generate investigative leads from a wide range of biological evidence.

Identification of the body fluid donor in mixtures through target mRNA cSNP sequencing.

In forensic practice, mixture stains containing various body fluids are common, presenting challenges for interpretation, particularly in multi-contributor mixtures. Traditional STR profiles face difficulties in such scenarios. Over recent years, RNA has emerged as a promising biomarker for body fluid identification, and mRNA polymorphism has shown excellent performance in identifying body fluid donors in previous studies. In this study, a massively parallel sequencing assay was developed, encompassing 202 coding region SNPs (cSNPs) from 45 body fluid/tissue-specific genes to identify both body fluid/tissue origin and the respective donors, including blood, saliva, semen, vaginal secretion, menstrual blood, and skin. The specificity was evaluated by examining the single-source body fluids/tissue and revealed that the same body fluid exhibited similar expression profiles and the tissue origin could be identified. For laboratory-generated mixtures containing 2-6 different components and mock case mixtures, the donor of each component could be successfully identified, except for the skin donor. The discriminatory power for all body fluids ranged from 0.997176329 (menstrual blood) to 0.99999999827 (blood). The concordance of DNA typing and mRNA typing for the cSNPs in this system was also validated. This cSNP typing system exhibits excellent performance in mixture deconvolution.

Evaluation of a New DNA Extraction Method on Challenging Bone Samples Recovered from a WWII Mass Grave.

Bones and teeth represent a common finding in ancient DNA studies and in forensic casework, even after a long burial. Genetic typing is the gold standard for the personal identification of skeletal remains, but there are two main factors involved in the successful DNA typing of such samples: (1) the set-up of an efficient DNA extraction method; (2) the identification of the most suitable skeletal element for the downstream genetic analyses. In this paper, a protocol based on the processing of 0.5 g of bone powder decalcified using Na2EDTA proved to be suitable for a semi-automated DNA extraction workflow using the Maxwell® FSC DNA IQ™ Casework Kit (Promega, Madison, WI, USA). The performance of this method in terms of DNA recovery and quality was compared with a full demineralisation extraction protocol based on Qiagen technology and kits. No statistically significant differences were scored according to the DNA recovery and DNA degradation index (p-values ≥ 0.176; r ≥ 0.907). This new DNA extraction protocol was applied to 88 bone samples (41 femurs, 19 petrous bones, 12 metacarpals and 16 molars) allegedly belonging to 27 World War II Italian soldiers found in a mass grave on the isle of Cres (Croatia). The results of the qPCR performed by the Quantifiler Human DNA Quantification kit showed values above the lowest Limit of Quantification (lLOQ; 23 pg/µL) for all petrous bones, whereas other bone types showed, in most cases, lower amounts of DNA. Replicate STR-CE analyses showed successful typing (that is, >12 markers) in all tests on the petrous bones, followed by the metacarpals (83.3%), femurs (52.2%) and teeth (20.0%). Full profiles (22/22 autosomal markers) were achieved mainly in the petrous bones (84.2%), followed by the metacarpals (41.7%). Stochastic amplification artefacts such as drop-outs or drop-ins occurred with a frequency of 1.9% in the petrous bones, whereas they were higher when the DNA recovered from other bone elements was amplified (up to 13.9% in the femurs). Overall, the results of this study confirm that petrous bone outperforms other bone elements in terms of the quantity and quality of the recovered DNA; for this reason, if available, it should always be preferred for genetic testing. In addition, our results highlight the need for accurate planning of the DVI operation, which should be carried out by a multi-disciplinary team, and the tricky issue of identifying other suitable skeletal elements for genetic testing. Overall, the results presented in this paper support the need to adopt preanalytical strategies positively related to the successful genetic testing of aged skeletal remains in order to reduce costs and the time of analysis.