Skeletal remains are the only biological material that remains after long periods; however, environmental conditions such as temperature, humidity, and pH affect DNA preservation, turning skeletal remains into a challenging sample for DNA laboratories. Sample selection is a key factor, and femur and tooth have been traditionally recommended as the best substrate of genetic material. Recently, petrous bone (cochlear area) has been suggested as a better option due to its DNA yield. This research aims to evaluate the efficiency of petrous bone compared to other cranium samples (tooth) and postcranial long bones (femur and tibia). A total amount of 88 samples were selected from 38 different individuals. The samples were extracted by using an organic extraction protocol, DNA quantification by Quantifiler Trio kit and amplified with GlobalFiler kit. Results show that petrous bone outperforms other bone remains in quantification data, yielding 15–30 times more DNA than the others. DNA profile data presented likeness between petrous bone and tooth regarding detected alleles; however, the amount of DNA extracted in petrous bones allowed us to obtain more informative DNA profiles with superior quality. In conclusion, petrous bone or teeth sampling is recommended if DNA typing is going to be performed with environmentally degraded skeletal remains.

Poor nuclear DNA preservation from highly degraded skeletal remains is the most limiting factor for the genetic identification of individuals. Mitochondrial DNA (mtDNA) typing, and especially of the control region (CR), using next‐generation sequencing (NGS), enables retrieval of valuable genetic information in forensic contexts where highly degraded human skeletal remains are the only source of genetic material. Currently, NGS commercial kits can type all mtDNA‐CR in fewer steps than the conventional Sanger technique. The PowerSeq CRM Nested System kit (Promega Corporation) employs a nested multiplex‐polymerase chain reaction (PCR) strategy to amplify and index all mtDNA‐CR in a single reaction. Our study analyzes the success of mtDNA‐CR typing of highly degraded human skeletons using the PowerSeq CRM Nested System kit. We used samples from 41 individuals from different time periods to test three protocols (M1, M2, and M3) based on modifications of PCR conditions. To analyze the detected variants, two bioinformatic procedures were compared: an in‐house pipeline and the GeneMarker HTS software. The results showed that many samples were not analyzed when the standard protocol (M1) was used. In contrast, the M3 protocol, which includes 35 PCR cycles and longer denaturation and extension steps, successfully recovered the mtDNA‐CR from highly degraded skeletal samples. Mixed base profiles and the percentage of damaged reads were both indicators of possible contamination and can provide better results if used together. Furthermore, our freely available in‐house pipeline can provide variants concordant with the forensic software.

In many human rights and criminal contexts, skeletal remains are often the only available samples, and they present a significant challenge for forensic DNA profiling due to DNA degradation. Ancient DNA methods, particularly capture hybridization enrichment, have been proposed for dealing with severely degraded bones, given their capacity to yield results in ancient remains. Background/Objectives: This paper aims to test the efficacy of genome-wide capture enrichment on degraded forensic human remains compared to autosomal STRs analysis. Methods: Six highly degraded human bones from the Spanish Civil War (1936–1939) were quantified with Quantifiler^™ Trio and amplified with GlobalFiler^™. Independently, partially UDG-treated double-stranded DNA libraries were generated and shotgun sequenced to screen for endogenous human DNA content. Subsequently, libraries were enriched with the Twist Bioscience “Twist Ancient DNA” reagent enrichment kit, which had not been previously tested for forensic purposes. Results: The results show that the samples behave similarly with both approaches (well-preserved samples yield good results). However, capture enrichment provides some new relevant insights, suggesting that its implementation in current NGS forensic platforms could be beneficial. Conclusions: Shotgun results show that the analyzed samples exhibit the same characteristics as ancient DNA samples in terms of DNA fragmentation and molecular damage, which may enhance the value of this approach when authenticating the endogenous DNA of forensic samples.

Background /Objectives: The sequencing of mitochondrial DNA is a valuable tool in forensic genetics, particularly in cases involving degraded samples or those with low nuclear DNA content. In this study, we performed an internal validation for an NGS-based typing of the mitochondrial DNA control region using the Precision ID mtDNA Control Region Panel on the Ion S5^TM sequencer (Thermo Fisher Scientific, Waltham, MA, USA). This validation enhances the scientific robustness, reliability, and judicial admissibility of the results in forensic cases. Methods: Six parameters were evaluated: minimum read depth, sensitivity, repeatability, concordance with Sanger, reproducibility and heteroplasmy detection employing ten negative controls, nine reference samples, a bone sample, and six experimental mixtures. Libraries were prepared using the Ion Chef^TM system, quantified on the Quantstudio^TM 5 Real-Time PCR, sequenced on the Ion GeneStudio^TM S5, and analyzed with Converge^TM software. Results: In this study, we found that a read depth threshold of 100 reads per position, an optimal concentration of 20 pg/µL, and a detection threshold of heteroplasmies of 20% are appropriate to obtain reliable genetic profiles. This supports the application of this method in forensic casework, in which initial concentrations may be around the optimal concentration exposed here due to the provenience of the samples. Conclusions: The results indicate that the NGS platform is suitable for forensic mtDNA analysis, even under low-template conditions, and offers higher sensitivity compared to Sanger sequencing. However, some limitations were observed in the coverage of specific amplicons, the detections of polymorphisms in homopolymeric regions, and in the detection of low-level heteroplasmies.

Age estimation using DNA methylation analysis is one of the tools currently used in forensic science to facilitate human identification. A total of 117 buccal swab samples from individuals aged 18 to 67 years were analyzed using a custom panel encompassing 610 CpG sites across 12 age-related genes. The analysis was performed using the amplicon-based technology Ion AmpliSeq™, which incorporates Ion Torrent S5 sequencing (Thermo Fisher Scientific, Waltham, MA, USA). The resulting methylation data were used to develop and compare age-prediction models based on Watson-strand, Crick-strand, and combined Watson + Crick methylation matrices. After candidate CpG selection and model reduction, the final Watson-strand model used 5 CpG sites from the age-related genes C1orf132, ELOVL2, KLF14, PDE4C and TRIM59 and was fitted using Elastic Net regression with alpha grid search, lambda selection and internal cross-validation. Model validation yielded prediction results with a mean absolute error of 4.42 years for the Watson matrix, 4.75 years for the Crick matrix, and 5.08 years for the WC matrix. These results allow for the evaluation of the suitability of using Ion AmpliSeq™ technology in forensic age estimation studies based on DNA methylation analysis.