Forensic microbiomics is a promising tool for crime investigation. Geolocation, which connects an individual to a certain place or location by microbiota, has been fairly well studied in the literature, and several applications have been found. The aim of this review is to highlight the main findings in this field, including the current sample storage, DNA extraction, sequencing and data analysis techniques that are being used, and its potential applications in human trafficking and ancient DNA studies. Second, the challenges and limitations of forensic microbiomics and geolocation are emphasised, providing recommendations for the establishment of this tool in the forensic science community.

Extracting DNA from degraded human remains poses a challenge for any forensic genetics laboratory, as it requires efficient high-throughput methods. While little research has compared different techniques, silica in suspension has been identified in the literature as the best method for recovering small fragments, which are often present in these types of samples. In this study, we tested five DNA extraction protocols on 25 different degraded skeletal remains. Including the humerus, ulna, tibia, femur, and petrous bone. The five protocols were organic extraction by phenol/chloroform/isoamyl alcohol, silica in suspension, High Pure Nucleic Acid Large Volume silica columns (Roche), InnoXtract™ Bone (InnoGenomics), and PrepFiler™ BTA with AutoMate™ Express robot (ThermoFisher). We analysed five DNA quantification parameters (small human target quantity, large human target quantity, human male target quantity, degradation index, and internal PCR control threshold), and five DNA profile parameters (number of alleles with peak height higher than analytic and stochastic threshold, average relative fluorescence units (RFU), heterozygous balance, and number of reportable loci) were analysed. Our results suggest that organic extraction by phenol/chloroform/isoamyl alcohol was the best performing method in terms of both quantification and DNA profile results. However, Roche silica columns were found to be the most efficient method.

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.