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- Forensic Evaluation of the Ion AmpliSeq MH-74 Microhaplotype Panel in the Portuguese PopulationPublication . Nascimento, Rui; Afonso Costa, Heloísa; Cunha, Eugénia; Amorim, AntónioBackground/Objectives: Microhaplotypes have emerged as promising genetic markers for forensic applications, combining low mutation rates with high polymorphic information. However, population data remain limited for several regions, including Portugal. This study aimed to generate population data for the Ion AmpliSeq™ MH-74 panel in a Portuguese population and evaluate its performance for forensic identification and kinship analysis. Methods: A total of 237 unrelated individuals were genotyped using massively parallel sequencing on the Ion GeneStudio S5 platform. Allele frequencies and forensic parameters were estimated. Population structure was assessed using principal component analysis (PCA) based on reference data from MicroHapDB. Kinship performance was evaluated through simulations of full siblings, half siblings, and first cousins and compared with STR markers using likelihood ratios (LR) and error rates. Results: The MH-74 panel showed high genetic diversity (mean He = 0.615; mean PIC = 0.565) and strong forensic efficiency, with very high combined power of discrimination and exclusion and a random match probability of 3.86 × 10−55. PCA revealed clustering consistent with global population structure, with the Portuguese population grouping within European populations. Microhaplotypes produced higher log10(LR) values for related pairs compared to STRs, particularly for closer relationships, with lower false negative rates. Conclusions: The MH-74 panel demonstrates high discriminatory power and reliable performance for kinship analysis in the Portuguese population, supporting the use of microhaplotypes as an effective complement to STR markers in forensic genetics.
- Forensic Genetics as a Tool for Peace and Justice: An Overview on DNA QuantificationPublication . Vieira Da Silva, Cláudia; Afonso Costa, Heloísa; Costa Santos, J.; Espinheira, RosaIn Forensic Genetics, DNA analysis is performed to obtain a Short Tandem Repeat (STR) profile from an evidence sample, which is then compared with the victim and suspect(s) reference sample STR profile, to determine their contribution to that evidence sample. However, forensic biological samples can be present in low quantities and be exposed to different environmental insults leading to DNA degradation and contamination by inhibitor compounds. Thus, it is desirable for a forensic scientist to have useful information about the forensic sample quantity and quality prior to STR amplification. New methods in Forensic DNA analysis for detecting, preserving, and quantifying DNA, as well as its recovery from different biological materials are continually being developed. Real-Time PCR (RT-PCR) assays for DNA quantification, like the recent Quantifiler® Duo DNA quantification kit (Applied Biosystems) proved to be very useful in forensic samples. Since many samples, mainly those resulting from sexual assault cases are often composed by unbalanced male/female DNA mixtures, the knew RT-PCR quantification assay, developed to quantify relative male/ female DNA ratio contributes not only to total DNA determination but also to ascertain the presence and quantity of enough male DNA in the sample. These results are important to guide the optimal STR analysis selection, such as autosomal STR, Y-STR, or mini-STR, increasing downstream analysis success rates. In this work we present real forensic casework where the DNA amount and quality were important to guide the selection of the appropriate STR amplification kit in order to increase the success of profiling in the first attempt, reducing the number of samples that need to be reprocessed and thereby decreasing the turn around time in a forensic laboratory.
- Genetic identification of human skeletal remains in forensic context: a reviewPublication . Cainé, Laura; Henriques, Madalena; Rohovska, Adelina; Sousa, Bárbara; Afonso Costa, Heloísa; Correia Dias, Maria Helena; Rodrigues, Joana; Franco, Magda; Mukan, Olena; Nascimento, Rui; Vânia Mofreita; Amorim, AntónioBackground/Objectives: Genetic identification of human skeletal remains plays a pivotal role in forensic investigations when other traditional or primary methods are not appropriate. Decomposition, storage and environmental conditions often leave the skeletal structure as the only basis for identification. This review synthesizes current methodologies and technological advances in damaged DNA extraction and analysis, emphasizing the forensic relevance of skeletal remains for genetic identification. Methods: A comprehensive literature analysis highlights the basis of genetic identification; sampling that considers intrinsic and extrinsic factors influencing the DNA yield and its quality; pre-treatment methods; extraction protocols that are suitable for its sensitivity; genetic marker panels that allow for human identification; and statistical evaluation and analysis of the results. The last chapter demonstrates the real-world impact of genetic identification on historical cases, underscoring its broader significance in legal, humanitarian, and socio-historical contexts, supporting a critical evaluation of best practices, methodological robustness, and ethical considerations within the field. Results: Teeth, femur and the petrous portion of temporal bone are the main samples used for genetic analysis. STR profiling and mitochondrial DNA are the gold standard markers for skeletal human identification. Minimally destructive protocols that enhance a high DNA yield are chosen, with silica-based methods being highlighted in the extraction protocols. Next-Generation Sequencing techniques have also improved analytical outcomes, by enabling high-throughput data generation, increased coverage depth, nucleotide-level sequence data, and high-level multiplexing of genetic targets. Conclusions: This review provides a comprehensive framework for researchers and practitioners seeking to optimize genetic identification workflows in forensic sciences and bioarcheology. These methodological advances have significantly increased identification success rates, especially in cases involving degraded or limited skeletal remains. Reviews such as this one help us to identify methodological gaps, ethical concerns, and future research directions, thereby establishing best practices when working with highly degraded skeletal material, supporting more reliable, standardized, and legally defensible applications of genetic identification in forensic, archeological, and humanitarian contexts.
- Human identification with combined anthropologic and genetic tools: two case reports in forensic medicine practicePublication . Amorim, António; Afonso Costa, Heloísa; Espinheira, Rosa; Costa, Rosa; Cunha, Eugénia; Costa Santos, JorgeThe identification of skeletonized corpses or skeletal remains, in forensic medicine practice, as well as being an imperative to human rights respect, has particular relevance and significance in psycho-social terms. If, on one hand, the Universal Declaration of Human Rights advises identification before all body inhumations, on the other hand, returning the identity to an unidentified corpse and subsequent return of the corpse to his family is, itself, the instrument which will allow for the possibility of beginning the family grieving. Forensic anthropology is the first approach towards the identification of unknown skeletonized corpses, which may lead to positive identification of the body or, sometimes, cannot go further than establish some pointers that will lead to further attempt to genetic identification. Here we report two cases of two dead bodies, unidentified, that were presented to the National Institute of Legal Medicine (INML), with instructions, from the prosecutors, to be determined possible cause and circumstances of death and achieve to individual identification. Forensic anthropology, allowed, in both cases, some indications that pointed to alleged missing individuals. Genetic study of skeletonized bodies and family members of the alleged missing individuals has turned possible, in one case, the positive identification. For one of the corpses, most likely due to the weather conditions to which he was exposed for a very considerable period, it was not possible to achieve to positive identification. However, in the latter case, we believe that the use of mitochondrial DNA study may help to settle the case with positive identification of the corps.
- mRNA profiling and donor association of mock casework samples: Results of a 3rd and 4th EDNAP collaborative exercisePublication . Hänggi, Nadescha Viviane; Amorim, António; Afonso Costa, Heloísa; Andersen, Jeppe Dyrberg; Kampmann, Marie-Louise; Courts, Cornelius; Neis, Maximilian; Syndercombe-Court, Denise; Giangasparo, Federica; Fonneløp, Ane Elida; Johannessen, Helen; Hadrys, Thorsten; Fürst, Angelika; Parson, Walther; Niederstätter, Harald; Sidstedt, Maja; Aili Fagerholm, Siri; Sijen, Titia; van den Berge, Margreet; Hanson, Erin; Ballantyne, Jack; Haas, CordulaSimultaneous identification and association of body fluids to donors can serve as a powerful tool in the criminal investigation of mixed traces. Massively parallel sequencing of mRNA targets not only identifies the origin of the body fluids but may also provide additional contextual information about the body fluid donors of a (binary) mixture using coding region SNPs (cSNPs). Within the European DNA Profiling Group (EDNAP), two consecutive collaborative exercises (3rd and 4th EDNAP exercise) were organized, with the objective to evaluate the performance of two previously published high-resolution mRNA sequencing assays. In the 3rd EDNAP exercise, the BFID-cSNP-BSS assay (cSNPs for blood, saliva, and semen) was evaluated, while in the 4th EDNAP exercise, the BFID-cSNP-6F assay (cSNPs for six fluids/tissues, including blood, saliva, semen, vaginal secretion, menstrual blood, and skin) was tested. Each RNA cSNP assay was accompanied by a genomic DNA assay for the genotyping of the cSNPs in the individual(s) or body fluid donor(s) of interest. A total of 11 laboratories participated in one or both collaborative exercises. In each exercise, the participating laboratories received a set of 16 standardized mock case stains for analysis and were encouraged to analyze additional, self-prepared stains and reference samples. Laboratories could participate using either the Ion Torrent S5™ or the Illumina MiSeq™ sequencing system. The results of the 16 mock case stains were very encouraging in both exercises, as body fluid components could be reliably identified for most of the stains. Since successful donor association depends on the number of body fluid markers covered in the sequencing results, we found that for stains consisting of blood, menstrual blood, vaginal secretion or a mixture thereof, the cSNPs provided substantial genetic discriminatory information for successful association of the respective body fluid to its donor. In mixtures, the difficulty in interpreting the cSNP genotypes might be attributed to the masking effect of the other body fluid(s) present. Body fluid identification and donor association of skin samples proved to be a significant challenge. In conclusion, body fluid identification and donor association using the BFID-cSNP-BSS and -6 F assays is a promising and effective method across laboratories and sequencing platforms.
- Quo vadis, BGA? A collaborative EDNAP exercise on the challenges and progress in forensic biogeographical ancestry inference.Publication . Diepenbroek, Marta; Amorim, António; Anslinger, Katja; Arora, Natasha; Amory, Christina; Ballard, David; Bekaert, Bram; Bouakaze, Caroline; Afonso Costa, Heloísa; Decorte, Ronny; Ewers, Lena; Fagerholm, Siri Aili; Garcia, Oscar; van der Gaag, Kris; Gysi, Mario; Haas, Cordula; Hollard, Clemence; Kling, Daniel; Palencia-Madrid, Leire; Pereira, Vania; de la Puente, María; Ruiz-Ramírez, Jorge; Sadam, Maarja; Sidstedt, Maja; Mogensen, Helle Smidt; Staadig, Adam; Stoljarova-Bibb, Monika; Court, Denise Syndercombe; Tillmar, Andreas; Tvedebrink, Torben; Xavier, Catarina; Weiler, Natalie; Parson, Walther; Phillips, ChristopherThere is a broad consensus that forensic tests for the prediction of externally visible characteristics (EVC) and analysis of biogeographic ancestry (BGA) of an individual are technically reliable. However, interpretation of the results and population-specific genotype distribution patterns remains challenging. EVC and BGA analyses provide valuable information for population genetics studies and as investigative leads for criminal cases, as well as for historical and contemporary identification tests. However, inaccurate or incorrect predictions, for example, from subjective bias in the interpretations made, have the potential to misdirect police investigations. The legal situation regarding EVC and BGA testing varies by country: ranging from countries where it is explicitly prohibited, to those without specific regulations on biogeographic ancestry prediction, and others that have already enacted laws governing its use. The reluctance to utilize these analyses is not only due to legal restrictions and data protection concerns, but also to initial limited sets of sufficiently comprehensive forensic DNA assays. Forensic BGA marker panels typically contain up to ∼300 SNPs. This relatively small number of genetic markers, along with limited reference population data, complicates the interpretation of results from donors of unknown origin. This paper presents the results of a collaborative EDNAP study, which, for the first time, evaluated the approach to reporting EVC and BGA data between international laboratories. For the study, DNA from nine individuals with self-reported ancestry was collected and analysed using various forensic panels differing in the number and composition of ancestry-informative markers genotyped, comprising: the Precision ID mtDNA Whole Genome Panel, the VISAGE Basic Tool and the VISAGE Enhanced Tool for Appearance and Ancestry Prediction, and the Ion AmpliSeq™ PhenoTrivium Panel. To ensure full data protection, all SNP genotypes and uniparental marker haplotypes obtained were not shared with third parties. Instead, the genetic data were analysed using a range of commonly used population analysis software packages. These analysis outcomes were then distributed to twelve European forensic laboratories (both academic and law enforcement institutions), who were asked to prepare reports based on their interpretation of the phenotypes and ancestry they inferred from the analysis data. A questionnaire sent alongside the genetic information, aimed to evaluate which difficulties were encountered by the participants in processing the BGA analysis data they were given.