The efficient extraction of DNA from challenging samples, such as bones, is critical for the success of downstream genotyping analysis in molecular genetic disciplines. Even though the ancient DNA community has developed several protocols targeting small DNA fragments that are typically present in decomposed or old specimens, only recently forensic geneticists have started to adopt those protocols. Here, we compare an ancient DNA extraction protocol (Dabney) with a bone extraction method (Loreille) typically used in forensics. Real-time quantitative PCR and forensically representative typing methods including fragment size analysis and sequencing were used to assess protocol performance. We used four bone samples of different age in replicates to study the effects of both extraction methods. Our results confirm Loreille’s overall increased gain of DNA when enough tissue is available and Dabney’s improved efficiency for retrieving shorter DNA fragments that is beneficial when highly degraded DNA is present. The results suggest that the choice of extraction method needs to be based on available sample, degradation state, and targeted genotyping method. We modified the Dabney protocol by pooling parallel lysates prior to purification to study gain and performance in single tube typing assays and found that up to six parallel lysates lead to an almost linear gain of extracted DNA. These data are promising for further forensic investigations as the adapted Dabney protocol combines increased sensitivity for degraded DNA with necessary total DNA amount for forensic applications.

Although not without subjectivity, the cranial trait scoring method is an easy visual method routinely used by forensic anthropologists in sex estimation. The revision presented by Walker in 2008 has introduced predictive models with good accuracies in the original populations. However, such models may lead to unsatisfactory performances when applied to populations that are different from the original. Therefore, this study aimed to test the sex predictive equations reported by Walker on a contemporary Italian population (177 individuals) in order to evaluate the reliability of the method and to identify potential sexual dimorphic differences between American and Italian individuals. In order to provide new reference data to be used by forensic experts dealing with human remains of modern/contemporary individuals from this geographical area, we designed logistic regression models specific to our population, whose accuracy was evaluated on a validation sample from the same population. In particular, we fitted logistic regression models for all possible combinations of the five cranial morphological traits (i.e., nuchal crest, mastoid process, orbital margin, glabella, and mental eminence). This approach provided a comprehensive set of population-specific equations that can be used in forensic contexts where crania might be retrieved with severe taphonomic damages, thus limiting the application of the method only to a few morphological features. The results proved once again that the effects of secular changes and biogeographic ancestry on sexual dimorphism of cranial morphological traits are remarkable, as highlighted by the low accuracy (from 56% to 78%) of the six Walker’s equations when applied to our female sample. Among our fitted models, the one including the glabella and mastoid process was the most accurate since these features are more sexually dimorphic in our population. Finally, our models proved to have high predictive performances in both training and validation samples, with accuracy percentages up to 91.7% for Italian females, which represents a significant success in minimizing the potential misclassifications in real forensic scenarios.

Bone taphonomy is a widely investigated topic; however, few data are available concerning marine bone taphonomy, especially on remains recovered from great depths and with short post-mortem intervals. To date, few studies have evaluated the bony changes which occur in seawater compared to samples with different post-mortem histories, and none through a comparative analysis of different approaches. To this purpose, this pilot study aims to examine the influence of seawater on bone preservation compared to other depositional contexts by multiple perspectives. Forty-nine human bone samples (femurs or tibiae) recovered from different environments (sea water, fresh water, outdoor, burial in coffin) were compared by macroscopic, microscopic and bone densitometric approaches. In order to investigate organic and inorganic components, undecalcified and decalcified histology of thin sections was performed. The analyses revealed a well-preserved bone tissue both macroscopically (92%) and microscopically (97% and 95% for undecalcified and decalcified sections). No significant differences were detected from radiological densitometric investigations (BMD = 1.6 g/cm² ± 0.1), except between old and young individuals (p value < 0.001). Differences were observed for body decomposition and few scavenged samples (3/15). However, even if slight variations were observed, no relation was recorded with the depositional contexts. We found a similar bone preservation in the four environments at the time of recovery, both macroscopically and microscopically, but also with radiological densitometric investigations. Our observations enriched the literature on bone taphonomy, providing data on bone tissue preservation in the early post-mortem period from a multidisciplinary perspective, paving the way for further studies on the topic.

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.

The identification of human fire victims is a challenging task in forensic medicine. The heat-induced alterations of biological tissues can make the conventional anthropological analyses difficult. Even if the DNA profile of the victim is achieved, it is possible that no match can be found in a forensic DNA database, thus hindering positive identification. In such cases, any information useful to nail down a possible identity should be collected, such as DNA methylation analysis which could provide useful investigative leads. In the present study, five age-related epigenetic markers (ELOVL2, FHL2, KLF14, C1orf132, and TRIM59) were initially analysed in blood samples of 72 living Italian individuals of known age, using a Single Base Extension (SBE) assay. An age prediction model was built by multiple linear regression including all the markers (Mean Absolute Error, MAE: 3.15 years). This model was tested on 29 blood samples collected during autopsies from burnt human remains, already identified through DNA analysis, providing a MAE of 6.92 years. The model allowed a correct prediction in 79.3% of the cases (95% prediction interval), while six cases were associated with inaccurate predictions (min-max prediction error: 9.8–37.3 years). Among the different sample variables considered to explain these results, only the DNA degradation index was a relevant factor affecting the reliability of the predictions. In conclusion, the SBE typing of blood from burnt remains proved to be a reliable tool to estimate chronological age of most of the samples, also in consideration of its cost-effectiveness and the availability of CE sequencers in every forensic genetics laboratory.