Percorrer por autor "Lopes, Virginia"
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- Analysis of paterniy cases with a single exclusion in a genetic marker using precision ID GLOBALFILER™ NGS STR PANEL v2Publication . Gouveia, Nair; Lopes, Virginia; Porto, Maria João; Bento, Ana; Andrade Sampaio, Lisa; Serra, Armando; Balsa, Filipa; Bogas, Vanessa; São Bento, Marta; Amorim, AntónioPaternity test results can sometimes evidence incompatibilities in the allelic transmission from parents to children, such as the presence of a single exclusion in one specific genetic marker, revealing a mismatch between the genetic profiles of the biological parent and the offspring. In these cases, it is important to determine whether the exclusion could be the result of a mutation or other factors as null or silent alleles. Capillary electrophoresis (CE) is the traditional method used in forensic genetics to analyze STRs (Short Tandem Repeats), however it is not possible to know the exact allele number variation due to the lack of sequence data. The application of Next-Generation Sequencing (NGS) technology may provide additional information, since it allows to detect and sequence simultaneously SNPs (Single Nucleotide Polymorphisms) present in the flanking regions and also distinguish isometric alleles with the same length but different sequences, that were misinterpreted as homozygous. In this study, a set of reference samples (buccal swabs and blood stains), previously amplified with the GlobalFiler™ PCR Amplification Kit and sequenced by CE on the 3500 Genetic Analyzer, were selected from paternity cases with a single exclusion, reported after GeneMapper ID-X Software analysis. All samples were automatically prepared with the Precision ID GlobalFiler™ NGS STR Panel v2 on the Ion Chef™ System, followed by sequencing on the Ion S5™ System and finally Converge™ Software analysis, according to the manufacturer’s instructions. The aim was to verify if the NGS methodology provides valuable information in these paternity cases and to identify the parental origin of a mutant allele. The NGS results were in concordance with those obtained by CE. In addition, this methodology demonstrated to be useful to clarify the paternity cases, because it enables a higher power of discrimination through 9 additional multi-allelic STRs, in a total of 35 markers instead of 24 markers of the GlobalFiler™ PCR Amplification Kit used in the traditional method. Therefore, the Precision ID GlobalFiler™ NGS STR Panel v2 shows to be a powerful method for kinship analyses and typing reference samples.
- A evolução e o impacto do SIIMP no SGBFPublication . Ferreira da Silva, Benedita; Lopes, Virginia; Afonso Costa, Heloísa; Amorim, AntónioO SIIMP revolucionou a forma de comunicação entre os diferentes serviços do INMLCF. Para cada serviço foi criado um módulo SIIMP adequado à sua atuação pericial. O SIIMP permitiu a introdução da política de ”processo único” o que o constituiu um benefício na transmissão, compilação e compreensão das informações e atuações de cada serviço em cada processo pericial. Os primeiros módulos do SIIMP: Genética, Toxicologia, Patologia e Anatomia Patológica, entraram em produção em novembro de 2023. O presente trabalho pretende mostrar a evolução do módulo do SGBF. Apesar da validação prévia, várias funcionalidades e permissões não se encontravam conforme o pretendido, pelo que correções e melhorias foram sendo implementadas à medida que iam sendo identificadas e reportadas. O SGBF iniciou o registo processual no SIIMP em novembro de 2023 e até janeiro de 2024 os registos foram realizados em duplicado, no SIIMP e StarLims, por forma a que pudessem ser identificados erros no seu funcionamento, sem prejuízo do trabalho pericial. Os relatórios e a faturação passaram a ser validados e enviados, diretamente do SIIMP, para o email da entidade requisitante, o que constituiu uma melhoria notável. Com a introdução do SIIMP no SGBF verificou-se um impacto significativo na organização processual de amostras provenientes da Patologia, com particular ênfase para o arquivo de manchas de sangue, deixando de ser criado novo registo com atribuição de processo SGBF. Também, em janeiro de 2024 com o módulo de Clínica a entrar em produção a interoperabilidade Genética-Clínica trouxe vantagens significativas, nomeadamente na transmissão de informação e indicações médicas relevantes, bem como o registo de todas as amostras colhidas, pelo perito médico, com as suas indicações e designações, sem necessidade de novo registo no SGBF. Em outubro de 2024 foi implementada outra funcionalidade com elevada potencialidade, a interoperabilidade CITIUS-SIIMP. Também esta funcionalidade necessitou de ajustes. O maior obstáculo encontrado relacionou-se com o facto de os serviços do Ministério Público e Tribunais criarem requisições diretamente no SIIMP. Muitas dessas requisições eram requisitadas a outro serviço do INMLCF, colocando em risco o devido seguimento no SGBF, por não serem conhecidas, o que implicou solicitações de transferência à RISI. Em 2025 o SIIMP torna-se o principal software de armazenamento dos documentos relacionados com as perícias médico legais, passando o EDOKLINK a ter um uso pontual. O SIIMP permitiu promover a uniformização do tratamento de toda a informação pericial não só entre o mesmo serviço em diferentes Delegações, assim como entre diferentes serviços médico legais e gabinetes médico legais, a nível nacional. No entanto, para que o SIIMP corresponda e permita que os profissionais do INMLCF beneficiem de toda a sua potencialidade, existem ainda lacunas na comunicação entre os diferentes serviços, que se pretende que possam ir sendo colmatadas.
- Internal Validation of the Investigator 26Plex QS Amplification Kit: a high-throughput multiplex assay for reference and low copy number DNA samplesPublication . Cardoso, Paula; Serra, Armando; Bogas, Vanessa; Balsa, Filipa; Lopes, Virginia; Dario, Rita; Porto, Maria João; Amorim, António; Corte Real, F.; Brito, PedroThe Investigator® 26plex QS Amplification Kit, from QIAGEN, provides reliable and rapid DNA profiles while enabling the multiplex amplification of 24 STRs, 2 Quality Sensors and a gender-determining marker, Amelogenin. The Quality Sensors incorporated in this kit provide insight into the sample quality and the PCR's success while alerting to the presence of inhibitors. Through an extensive internal validation study, this research sought to implement the Investigator® 26plex QS in the laboratory routine of the Forensic Genetic and Biology Service, Central branch (SGBF-C) of the Portuguese National Institute of Legal Medicine and Forensic Sciences. Here we detail the procedures and parameters applied which followed the SWGDAM guidelines, as well as report the results obtained throughout. Firstly, it was crucial to establish unique analytical criteria that would be the baseline for the interpretation of the results obtained. To accomplish such, analytical, stochastic, heterozygous balance and stutter thresholds were defined. Thereupon, this study intended to gauge the kit’s performance, by evaluating its concordance with normalized methodologies while assessing its sensitivity, specificity, robustness, and precision, besides its efficiency in the presence of degraded and/or inhibited samples. Lastly, in favour of optimizing the laboratory workflow, an assay was carried out to test half volume reactions and direct amplification on reference samples. In this study a wide range of sample types were analysed, which made it possible to acquire robust data pertaining to the performance of the assay. The laboratory methodology applied comprehended: DNA extraction using Prep-n-Go™ Buffer and PrepFiler Express™ Forensic DNA Extraction Kit, quantification with the Quantifiler™ Trio Quantification Kit, followed by the amplification with the Investigator® 26plex QS. Capillary electrophoresis was performed in the Applied Biosystems™ 3500 Genetic Analyzer, and the electropherograms’ were analysed using the GeneMapper™ ID-X Software v1.6. Through this work, it was possible to characterize the main advantages of the Investigator® 26plex QS kit, as well as its limitations. The validation data demonstrated that this system produces reliable profiles in the presence of minute DNA quantities and identifies the minor contributor in a mixture up to a 1:50 ratio. The results inferred a high human specificity, robustness, and sensitivity, while producing concordant and reproducible results with optimized protocols for both reference and low copy number DNA samples. Through concordance studies it was further shown that there is a high consistency between this novel amplification kit and those currently implemented in the SGBF-C, thus proving its suitability for the analysis of forensic samples.
- Internal Validation of the Investigator 26Plex QS Amplification Kit: a high-throughput multiplex assay for reference and low copy number DNA samplesPublication . Neves Cardoso, Paula Liliana; Serra, Armando; Bogas, Vanessa; Balsa, Filipa; Lopes, Virginia; Dario, Rita; Porto, Maria João; Amorim, António; Corte Real, Francisco; Brito, PedroHuman singularity provides a foundation for forensic genetics, where the study of Short Tandem Repeats (STRs) is key. Furthermore, the development of polymerase chain reaction (PCR) has revolutionized the field by enabling the amplification of specific DNA markers. The Investigator® 26plex QS Amplification Kit is a highly robust multiplex PCR system, for human identification. In this study, we report the results obtained from the internal validation carried out at the Portuguese National Institute of Legal Medicine and Forensic Sciences. The results showed that the Investigator® 26plex QS kit proved to be a robust tool for the analysis of forensic samples, offering high sensitivity and specificity.
- Reverse body fluid identification workflow: a direct to DNA ApproachPublication . Porto, Maria João; Ferreira, Joana; Bento, Ana Margarida; Bogas, Vanessa; Lopes, Virginia; Sampaio, Lisa; Gouveia, Nair; Corte Real Gonçalves, FranciscoWhen forensic DNA laboratories receive evidence from a crime scene their first task is to check for the presence of biological material, namely blood, semen or saliva; the same principle is applied to clothing or swabs related to victims from sexual assault cases. Examination of the exhibits by naked eye or using a forensic light source is done in order to detect the presence of body fluid stains. Many laboratories perform preliminary tests on items where biological material is potentially present before sending a cutting or swab for extraction and subsequent DNA typing. To identify the presence of body fluids our laboratory has implemented presumptive and/or confirmatory assays to detect semen, blood and saliva and, until the end of 2022, all samples selected for DNA extraction and posterior amplification were also tested to determine the type of biological evidence in question (whenever enough sample was available) in two independent workflows. For semen identification, all presumptive positive results were then tested in order to visualize sperm cells. However, in sexual assault cases there are many samples with a semen presumptive positive result but with a negative confirmatory test, meaning that this biological fluid cannot be confirmed. On the other hand, it was detected that in several situations the analysed samples did not present probative DNA results and, consequently, it would not have been necessary to test them for the presence of bodily fluids. The aim of this study was to propose a more efficient workflow to be applied to all forensic samples 647 samples from sexual assault crimes (male/female victims), occurred between 2020 and 2021, were selected and the results obtained in both previously mentioned workflows were evaluated and compared with a Direct to DNA approach, in which DNA analysis is performed prior to body fluids identification (carried out only on samples with eligible DNA profiles for the criminal case under study). The results revealed that if a Direct to DNA approach was adopted, only 256 samples (39,6%) would have been tested for the presence of semen (confirmed in 80 samples). Therefore, this workflow (currently implemented in our laboratory) is less laborious and time-consuming, allows standardization of the techniques implemented and, above all, no loss of information relevant to the judicial process was detected