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- Insertion/Delection Polymorphism and forensic aplications: A preliminary studyPublication . Vieira Da Silva, Cláudia; Matos, Sara; Amorim, António; Afonso Costa, Heloísa; Morais, Paulo; Santos, Rodolfo; Espinheira, Rosa; Santos, J. CostaThe human genetic identification is usually based on the study of STR markers, robust and reliable for samples containing relatively small quantities of DNA. Recent advances in forensic genetics have focused on the development of genotyping assays using shorter amplicons, in order to improve the successful amplification of degraded samples. Single Nucleotide Polymorphisms (SNP) and Insertion/Deletion polymorphisms (INDEL), length polymorphisms created by insertions or deletions of one or more nucleotides in the genome, have considerable potential in this kind of forensic samples, usually present in identification casework, since they can combine desirable characteristics of both, STR and SNP. In this study, a set of 30 biallelic Deletion/Insertion polymorphisms (DIP or INDEL) distributed over 19 autosomes plus Amelogenin in a single multiplex PCR reaction was applied to 100 healthy and unrelated caucasian individuals. Statistical analysis revealed that the 30 biallelic markers can provide satisfactory levels of informativeness for forensic demands.
- Forensic genetic analysis of South Portuguese population with the six dye Powerplex® Fusion 6CPublication . Vieira Da Silva, Cláudia; Afonso Costa, Heloísa; Porto, Maria João; Cunha, E; Corte Real, F.; Amorim, AntónioAs an improvement in efficiency and in Human Discrimination Power, the new six dye multiplex kit PowerPlex® Fusion 6C System, by Promega, available for human identification can co-amplify 27 loci, in a single reaction, have been introduced in the last years with great success. This kit allows the amplification and detection of autosomal loci included in the expanded Combined DNA Index System CODIS, plus the loci Penta D, PENTA E and SE33 as well as Amelogenin for gender determination. Furthermore, this kit includes three Y –STRs (DYS391, DYS576 and DYS570), allowing allelic attribution in a total of 27 loci. This genetic markers extension satisfies not only CODIS but also European Standard Set recommendations. Thinking about continuous human migration movements, especially in a very cosmopolitan region like Lisbon and south of Portugal, and also, in keeping population studies and actualized STR databases we decided to update our previous studies. Our sample is composed of 600 unrelated individuals, from paternity testing with laboratory identity anonymised. DNA was extracted by Prep-n-go BufferTM(Thermo-Fisher Scientific). PCR amplification was performed with PowerPlex® Fusion 6C System, according to manufacturer’s guidelines. Fragment analysis was carried out in an Applied Biosystems® 3500 Genetic Analyser. Electrophoresis results were analysed with GeneMapper® ID-X v1.4. Allele frequencies and population statistics, including Hardy-Weinberg equilibrium p-values from exact test probabilities and forensic parameters were calculated with adequate software. In conclusion, our population information was updated in order to apply most recent data in our casework weight of evidence.
- Population Genetic Data for F13A01, FES/FPS, F13B and LPL in the South Portuguese PopulationPublication . Vieira da Silva, Cláudia; Amorim, António; Afonso Costa, Heloísa; Espinheira, Rosa; Costa Santos, JorgeDNA parentage testing is currently performed using several highly polymorphic short tandem repeats (STRs). In our routine casework, we apply two validated STRs kits, in order to have results in the 13 codis loci plus D2S1338, D19S433, PENTA E, PENTA D, and Amelogenin. In complex and deficient paternity cases it is often necessary to increment the number of studied STRs. For this reason, we introduced in our laboratory GenePrint® FFFL Multiplex kit, which can provide results in F13A1, FES/FPS, F13B, and LPL using the GenePrint® FFFL System (Promega, USA) kit. In this study, we analyzed 150 unrelated and healthy individuals from the south Portugal population. Allele frequencies and statistical parameters were estimated with Arlequin 3.5.1.2. Paternity Statistics were calculated using software package PowerStats v12. The forensic efficiency values suggested that loci F13A01, FES/FPS, F13B, and LPL are discriminative and very useful to solve complex forensic casework, and should be added to the set of STRs loci routinely used in Forensic laboratories. In conclusion, an additional 4 loci dataset was established for the south Portuguese population, which can be used for both forensic casework and complex kinship testing
- QUANTIFILER®TRIO DNA method performance in a collection of ancient samplesPublication . Vieira- Silva, C.; Lopes, J.; Afonso Costa, H.; Ribeiro, T.; Porto, M.J.; Dias, M; Cunha, E; Amorim, A,During the past few years significant progress has been made in solving technical challenges associated with STR profiling including the ability to analyze degraded DNA and low amounts of DNA. The result of these changes is that useful STR profiles can now be obtained from previously untypeable forensic DNA samples. Analysis of DNA from ancient material represents an important role in molecular anthropology, although there are many limitations concerning low DNA quantity and/or degraded DNA, and/or PCR inhibitors. These factors can make it difficult to decide whether to continue with STR analysis, which STR panel to use and how much DNA to add to PCR reaction. With all these constraints, DNA quantification represents an important tool to decide which method will follow in order to improve workflow and have good results in less time-consuming. The Quantifiler® Trio DNA method provides a quality index (QI) to detect the presence of degraded DNA along with PCR inhibitors.This guide allows the selection of the optimal short tandem repeat (STR) analysis chemistry (autosomal, or miniSTR) and streamlines the workflow while increasing downstream analysis success rates. In order to compare DNA quality from different extraction methods, samples from 46 exhumed Middle Ages individuals were extracted with modified phenol-chloroform method and also PrepFiler Express BTA™ method. DNA was quantified with Quantifiler® Trio DNA Quantification in an Applied Biosystems® 7500 Real-Time PCR System. Results were analyzed and allow us to point Quantifiler® Trio method as an important tool in pre-STR typing methods in ancient samples
- Mitochondrial DNA in human identification: a reviewPublication . Amorim, António; Fernandes, Teresa; Taveira, NunoMitochondrial DNA (mtDNA) presents several characteristics useful for forensic studies, especially related to the lack of recombination, to a high copy number, and to matrilineal inheritance. mtDNA typing based on sequences of the control region or full genomic sequences analysis is used to analyze a variety of forensic samples such as old bones, teeth and hair, as well as other biological samples where the DNA content is low. Evaluation and reporting of the results requires careful consideration of biological issues as well as other issues such as nomenclature and reference population databases. In this work we review mitochondrial DNA profiling methods used for human identification and present their use in the main cases of humanidentification focusing on the most relevant issues for forensics.
- Y-Filer Plus® genetic characterization of caucasian individuals from South PortugalPublication . Vieira da Silva, Cláudia; Afonso Costa, Heloísa; Proença, Marta; Ribeiro, Teresa; Porto, Maria João; Amorim, AntónioDue to their paternal inheritance, Y-STRs offers particular perspectives for identification and kinship analysis and are also a precious tool in sexual assault cases with relatively high amount of female DNA and also in mixtures from multiple male donors. Nonetheless their value, there are some limitations to their use in forensic investigations since their ability to discriminate between individuals is considerably lower than that of the autosomal STRs set, mainly in cases with close or distant patrilineal relatives.One of the most recently developed Y-STR kit, Y-Filer Plus® (Life Technologies, Foster city, USA), allows forensic geneticists to study 27 Y-chromosomal loci. All the 16 markers included in the Y-Filer® kit (Life Technologies, Foster city, USA), plus 9 additional markers: DYS576, DYS627, DYS460, DYS518, DYS570, DYS449, DYS481, DYF387S1 and DYS533, six of which (DYS576, DYS627, DYS518, DYS570, DYS449 and DYF387S1) are characterized as “rapidly mutating”, and can differentiate between unrelated individuals and possibly between male relatives.Allelic frequencies were estimated with Arlequin v. 3.5. Gene and Haplotype diversities were estimated according to Nei formula. The discrimination capacity was also calculated by dividing the number of different haplotypes by the total number of individuals in the sample. The fraction of unique haplotypes was determined as the percent proportion of unique haplotypes. In conclusion, the recently introduced Y-Filer Plus® system provides innovative discriminatory power for forensic application
- El papel de las Ciencias Forenses en la identificación de restos óseos - informe de un caso (poster)Publication . Costa Lopes, Miguel; Abreu, Ana; Amorim, António; Ribeiro, Teresa; Cunha, Eugénia; Eiras, Luísa
- Pesquisa de SARS-CoV-2 em cadáveres: experiência da Delegação do Sul do Instituto Nacional de Medicina Legal e Ciências ForensesPublication . Logrado, Diana; Inácio, Ana Rita; Amorim, António; Santos, Carlos dos; Cunha, E
- Implementação da deteção molecular de SARS-CoV-2 na Delegação do Norte do Instituto Nacional de Medicina Legal e Ciências ForensesPublication . Fadoni, Jennifer; Magalhães, Jéssica; Cerqueira, Joana; Cainé, Laura; Amorim, AntónioA Doença do Coronavírus 2019, causada pelo vírus SARS-CoV-2, foi classificada como uma pandemia pela OMS em 11 de março de 2020. Até julho de 2023, foram registados mais de 768 milhões de casos, com mais de 6,9 milhões de mortes associadas. O objetivo do Laboratório de Virologia e de Análises Clínicas e Forenses da Delegação do Norte (LVACF-N) foi implementar a análise de deteção molecular de SARS-CoV-2 na Delegação do Norte do INMLCF, pois a deteção de casos de infeção por SARS-CoV-2 é de extrema importância para controlar a transmissão da infeção, o que por sua vez permite minimizar o impacto da COVID-19 na população e na demanda dos serviços públicos de saúde. A implementação da deteção molecular de SARS-CoV-2 na Delegação do Norte foi dividida em três etapas: i) Validação da metodologia de análise no equipamento de RT-PCR presente na Delegação do Norte, utilizando primers desenhados in house; ii) Avaliação do limiar de deteção do genoma viral; e iii) Validação do novo equipamento de RT-PCR adquirido para o LVACF-N, utilizando um kit comercial e também primers desenhados in house. Na fase I, foram realizadas análises de deteção molecular de SARS-CoV-2 em 61 zaragatoas nasofaríngeas colhidas post-mortem, utilizando primers desenhados in house. Os resultados foram comparados aos resultados de uma entidade externa, e foram obtidos valores de sensibilidade e especificidade iguais a 100%. Na fase II, foi possível detetar o genoma viral com uma concentração ínfima de 0,25 cópias/uL. Na fase III, foi avaliada a reprodutibilidade da análise no RT-PCR CFX96™ System em 30 amostras, utilizando primers desenhados in house e um kit comercial. Os resultados foram comparados aos resultados do equipamento validado na fase I, e não foi encontrada nenhuma divergência. Após a validação dos métodos e dos equipamentos, as análises de deteção molecular de SARSCoV- 2 em amostras colhidas post-mortem pelo Serviço de Clínica e Patologia Forense da Delegação do Norte, passaram a ser realizadas pelo LVACF-N. Entre 31/03/2023 e 21/07/2023, foram processadas 261 amostras, das quais 245 (93,87%) apresentaram resultado “negativo”, 12 (4,60%) “positivo” e 4 (1,53%) “indeterminável”; 177 (67,82%) foram colhidas em cadáveres do sexo masculino, 82 (31,42%) do sexo feminino, e 2 (0,76%) em cadáveres cujo sexo não foi identificado. A média de idade dos indivíduos foi de 63,25 anos, com valores entre 2 e 96 anos de idade. Em um curto intervalo de tempo, o LVACF-N validou metodologias e equipamentos, o que permitiu que as análises de deteção molecular de SARSCoV- 2 em amostras colhidas post-mortem pelo Serviço de Clínica e Patologia Forense desta Delegação, passassem a ser processadas internamente. Assim sendo, o LVACF-N aumentou a capacidade de autossuficiência do INMLCF, reduziu custos, possibilitou um maior aproveitamento de recursos, e pavimentou o caminho para a realização de outros tipos de análises de deteção molecular, a serem realizadas pelo LVACF-N num futuro próximo.
- In house Real-Time PCR multiplex for simultaneous detection of human influenza A and B virus and respiratory syncytial virusPublication . Fadoni, Jennifer; Cainé, Laura; Rodrigues, Joana; Mofreita, Vânia; Nascimento, Rui; Mukan, Olena; Corte Real, F.; Amorim, António