Aplicações da tecnologia de sequenciamento massivo paralelo em exames genéticos de interesse forense : alternativas analíticas para a casuística brasileira

• Autor(a) principal: Silva, Eduardo Filipe Avila
• Data de Publicação: 2019
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: Biblioteca Digital de Teses e Dissertações da PUC_RS
• Texto Completo: http://tede2.pucrs.br/tede2/handle/tede/9025
• topic: Genética Forense; Sequenciamento Massivo Paralelo; População Brasileira; Marcadores SNP; DNA Mitocondrial; Cromossomo Y; CIENCIAS BIOLOGICAS::BIOLOGIA GERAL

Molecular biology techniques constitute a significant part of technical solutions available in forensic analysis for criminal or civil purposes. As such, these methods are particularly relevant in cases including crime suspects identification, mass disaster victim identification, paternity or other forms of kinship determination, among others. In this context, forensic analysis usually faces technical problems associated with the very nature of criminal biological evidence: samples with very low DNA amounts or in highly degraded conditions (usually due to environmental exposure), DNA mixtures including more than a single origin, background DNA contamination, inhibitors presence, and additional factors. This situation might represent a challenge to the analysts, where alternative technologies to traditional methods can be the only alternative for successful evaluation of some criminal evidences. Massive Parallel Sequencing (MPS) techniques present advantages in challenging samples processing including high sensitivity, flexibility for panel design including different kind of forensic markers and polymorphisms, adequacy for use with degraded or contaminated samples, simultaneous processing of a high number of samples and/or polymorphisms (as a result of barcoding alternatives and humongous amount of data generated in a single run), along with other benefits. Therefore, inclusion of these techniques to routine analytical repertoire is key to efficient solution of current forensic problems caused by limits associated with traditional DNA genotyping, sequencing and analysis methods. Adequacy and convenience of MPS techniques introduction and implementation in Brazilian Federal Police Forensic Genetics Regional Laboratory were evaluated in this project, in order to add such methods to available DNA analysis protocols in this facility. Genotyping efficiency studies were conducted in a commercial MPS panel including 90 highly heterozygous, informative SNP markers, as well as 34 high-clade Y-chromosome SNPs. Allelic frequencies for Brazilian populations and other relevant forensic descriptive indexes were estimated. In addition, efficiency of this method in real criminal casework samples was evaluated, and results compared to those obtained from traditional analysis methods. Impact of biological nature of criminal samples in genotyping efficiency was also analyzed. Finally, genetic variability and structure in Y-chromosome haplotypes distributions was assessed in Brazilian population, according to geopolitical regions, in order to describe paternal lineage contribution patterns to modern Brazilian populations. Over four hundred reference or criminal samples from all five Brazilian geopolitical regions were successfully evaluated, and resulting allele frequencies and forensic parameters obtained for human identification purposes. A second commercial solution was also evaluated in its efficiency to generate complete DNA sequences of forensic relevant uniparental lineages. Thus, complete mitochondrial genomes were obtained through MPS techniques, whose efficiency and adequacy to Brazilian samples were explored. This study aims to lay the groundwork of a future database containing full-genome mitochondrial Brazilian sequences, for forensic purposes. Obtained results suggest adequacy of evaluated methods, which were found to be appropriate for routine use in Brazilian forensic casework processing, with ability to generate robust, efficient and trustworthy genetic profiles from Brazilian criminal samples.