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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
- Investigação de parentesco biológico: a importância de marcadores adicionais em casos de especial complexidadePublication . Rodrigues, Diogo; Vieira Da Silva, Cláudia; Carvalho, Mónica; Afonso Costa, Heloísa; Sampaio, Lisa; Cunha, E; Corte Real, F.; Amorim, AntónioA grande maioria das perícias de investigação de parentesco biológico realizadas pelo Instituto Nacional de Medicina Legal e Ciências Forenses (INMLCF) têm inicio com a ordem do Tribunal para realização da mesma. O mais frequente é o Tribunal dar ordem para nos serem presentes, como intervenientes, um trio constituído por um suposto pai, uma mãe e um(a) filho(a), havendo, no entanto, variações quanto ao número ou tipo de intervenientes, o que pode resultar em maior dificuldade em apresentar resultados com a robustez desejada. Em qualquer dos casos, genericamente, as conclusões possíveis de um estudo de parentesco biológico e mais concretamente de um estudo de paternidade são a exclusão ou não exclusão de paternidade relativamente ao suposto pai em estudo. A conclusão pela não exclusão é sempre acompanhada pela valorização estatística dos resultados, designadamente através do cálculo e apresentação do Índice de Parentesco (IP) e da Probabilidade de Parentesco (W). No caso de uma perícia de investigação de parentesco em que nos seja presente unicamente um filho biológico do suposto pai e um suposto filho biológico, sem possibilidade de estudo das amostras das respetivas mães biológicas nem da amostra biológica do suposto pai, e nos é pedido o estudo sobre a possibilidade de ambos serem filhos biológicos do mesmo pai, a impossibilidade de exclusão da paternidade pode estar associada a valores calculados de IP que podem ser pouco robustos. O ensaio Investigator® HDplex permite o estudo de marcadores genéticos adicionais aos habitualmente estudados na rotina pericial do INMLCF.
- The role of DNA concentrations in forensic casework results : regression models applicationPublication . Vieira Da Silva, Cláudia; Amorim, António; Afonso Costa, Heloísa; Porto, Maria João; Corte Real, F.; Antunes, MariliaIn forensic DNA typing, short tandem repeats (STRs) are the most frequently genotyped markers in order to distinguish between individuals and to relate them to a crime or to exonerate the innocent. In recent years, new controversies have arisen with the advent of more sensitive techniques, allowing profiles to be recovered from minimum amounts of DNA, hence, bringing challenges to weight of evidence evaluation for forensic DNA profiles obtained from low template DNA samples. Introduction of interpretation models, or even new weight of evidence software should be accompanied by a measure of uncertainty that is part of any biological analysis. Specially, due to stochastic effects, the reliability of the obtained profiles might differ between machinery, workflow and also PCR settings in use in different laboratories. In this work we try to understand the relation between Peak Area, DNA concentration and also size marker, using adequate regression models. Buccal swabs from 180 individuals, with unknown identity, were selected for this study. DNA was extracted with prep-n-go™ buffer and quantified using Quantifiler® Trio DNA Quantification kit in a 7500 Real-Time PCR System (Applied Biosystems). STR amplification was performed with Powerplex Fusion 6C amplification kit (Promega). Amplified PCR products were separated and detected in an Applied Biosystems® 3500 Genetic Analyzer using manufacturer’s conditions. Electrophoresis results were analysed with GeneMapper® ID-X v1.4. Statistical analysis was performed with R Studio. Our results allow having an important overview about the relation between DNA concentrations, peak area, and size of the studied genetic markers.
- 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.
- 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
- Variabilidade da quantidade de ADN em zaragatoas bucais –estudo preliminarPublication . Lucas, Isabel; Oliveira, Rita; Dourado, Catarina; Dario, Paulo; Reis, R.; Vieira Da Silva, Cláudia; Amorim, António; Afonso Costa, Heloísa; Simão, F.; Ribeiro, Teresa; Porto, Maria João; Costa Santos, JorgeOs laboratórios de Genética Forense têm como objetivo a obtenção de perfis genéticos com vista à identificação humana, para a resolução de perícias do âmbito cível e criminal. Com vista à obtenção dos perfis genéticos dos indivíduos, é necessário efetuar colheitas de amostras biológicas dos mesmos, denominadas por amostras de referência. As mais comummente usadas são as obtidas por descamação do epitélio da mucosa bucal, com recurso ao uso de zaragatoas bucais. A variabilidade da quantidade de células colhidas através deste procedimento, pode ser originada por diversos fatores. O objetivo deste estudo é avaliar fatores que eventualmente possam contribuir para tal facto.
- Lisbon population mtDNA analysis: study of a genetic marker with population, evolution and forensic interestPublication . Afonso Costa, Heloísa; Amorim, António; Vieira da Silva, Cláudia; Porto, Maria João; Cunha, Eugénia; Lemos, Manuel Carlos; Corte Real, FranciscoMitochondrial DNA mtDNA is remarkable in population genetic studies to clarify the history and demographic past of human populations For forensic purposes mtDNA analysis is particularly useful when there are degraded or low-level DNA samples Nonetheless, only being in possession of the genetic reference data of the populations it is possible to attribute statistical weight to evidence in forensic casework.The National Institute of Legal Medicine and Forensic Sciences in Portugal carried out a study with the aim of portraying the genetic diversity of immigrants living in Lisbon However, the nonimmigrant population of Lisbon, the reference population, had not yet been studied The present study intends to determine the mtDNA variability of the Lisbon metropolitan native population. Among the 90 samples, 87 unique mtDNA haplotypes were identified It was verified that 32 of the haplotypes had no coincidence and 14 of the haplotype had only one coincidence on EMPOP database This low frequency of the majority of the haplotypes emphasizes the importance of studying this population The most frequent haplotypes, enclose haplogroups H 1 HV, K 1 R 0, and U 5 belonging predominantly to west Eurasian mtDNA Haplotypes that belong to Haplogroups L 0 L 1 L 2 L 3 were also identified in Lisbon population.
- Mitochondrial DNA data of Cabo Verde Immigrant Population Living in LisboaPublication . Afonso Costa, Heloísa; Morais, P.; Amorim, António; Vieira da Silva, Cláudia; Matos, S.; Marques Santos, R.; Espinheira, R.; Costa Santos, J.Mitochondrial DNA (mtDNA) analysis found an important role in forensic genetics, especially when nuclear DNA analysis does not give a conclusive response. It is a powerful tool to exclude samples as originating from the same matriline. Features that increase the vested interest of mtDNA are the high copy number per cell, maternal inheritance, absence of recombination, and high mutation rate. Due to the higher overall mutation rate, the control region is comparatively enriched in sequence variation and therefore its analysis is important to establish haplotypes and haplogroups. Haplogroup assignment became noteworthy to clarify the history and demographic past of a population. As well as occurring all over Europe, in Portugal, and particularly in Lisboa, immigrant populations are increasing. The Instituto Nacional de Medicina Legal e Ciências Forenses is carrying out a comprehensive genetic study with the aim of portraying the genetic diversity of the immigrants who live in Lisboa. Within that objective, the present study intends to: obtain the mtDNA variability of Cabo Verde Immigrant Population Living in Lisboa and classify haplotypes into haplogroups. The studied population shows great interpopulation genetic variability due to the high frequency of unique haplotypes. Cabo Verde immigrants living in Lisboa exhibit haplotypes that belong to haplogroups observed in native Africans and in West Eurasian. MtDNA control region typing is extremely useful as a technique to differentiate among degraded samples frequently found in forensic genetics and to establish its global frequency when having knowledge of the genetic structure of populations.