Soluções para problemáticas forenses da Cannabis sp. no Brasil com foco genético e genômico

Abstract

The Cannabis sativa, known as Cannabis, is a plant shrouded in controversy due to divergent global legislations ranging from legalization to prohibition. Since its initial regulation in 1925, its growing economic and medicinal interest has influenced laws in many countries. According to UNODC, Cannabis remains the most globally consumed drug, with over 4% of the world population using it in 2020. This increased demand contributes to a highly lucrative market, associated with large-scale organized crime. Brazil, facing challenges in combating trafficking due to its extensive border and remote areas, seeks strategies, including UNODC molecular methods, for the identification and combat of Cannabis trafficking. Drug trafficking, especially of Cannabis, poses a global challenge, being even more complex in Brazil, where 7.7% of the population has experimented with the substance. The massive drug seizures in 2020 highlight the magnitude of the problem, complicated by diverse trafficking routes. Brazilian studies using STR panels show efficacy, offering a promising approach for tracking and identifying samples. The absence of population studies on the genome of trafficked plants in Brazil is a hurdle to the development of specific molecular tools, hindering investigations and legal processes. Genetic tests can provide accurate data for identification, qualification, and population inference, but logistical and technological obstacles persist in the Brazilian context. The lack of international molecular comparison and the shortage of expertise and equipment in Brazilian forensic laboratories accentuate the challenges in implementing these tools. To positively contribute to the fight against drug trafficking, this work developed three solutions. To address the issue of marijuana seed trafficking, a specific molecular analysis protocol for cannabis seeds was developed and validated. The study involved the extraction and genotyping of 43 seeds using a carefully developed protocol to ensure DNA integrity. DNA quantification was performed using the Qubit dsDNA HS Assay Kit. A multiplex assay was employed, focusing on 13 Short Tandem Repeat (STR) markers specific to Cannabis sativa, followed by PCR amplification and capillary electrophoresis for genotyping. Validation steps included a sensitivity assessment, species specificity tests, and reproducibility evaluations using Humulus lupulus seeds and Homo sapiens samples. Forensic statistical analyses were conducted to assess marker suitability, and concordance analyses were performed using a separate multiplex STR panel. Population assignment and genetic variability analyses were conducted using a dataset from drug trafficking routes. This part of the work resulted in a unique study, developing a robust methodology for forensic analysis, ensuring the reliability and applicability of the protocols developed to address the challenges of Cannabis sativa seed trafficking in Brazil. In the second experiment of the thesis, 45 samples of Cannabis sativa, representing four main origins, were selected for complete genome sequencing. The selection was based on DNA concentration, quality of XII 13-STR multiplex panel results, and the genotype of the 19 evaluated markers. Sequencing was performed using the Illumina NovaSeq protocol, resulting in coverage exceeding 8x for all samples. The data obtained, after alignment with the can10 reference genome, will be made available online to contribute to genetic research. Additionally, a population genetic database was created, using two internationally validated 13-STR multiplex panels. This database includes allelic frequencies and forensic parameters for the 19 markers, serving as a valuable tool for population inference analyses in forensic science. The results indicate that eight of the 19 markers showed deviation from Hardy-Weinberg equilibrium, justifiable by specific selection for origin differentiation. The combined discrimination power was estimated at 1 in 59 million. The database and forensic parameters provide substantial resources for routine application in police analyses, strengthening the fight against drug trafficking. In the third experiment, SNP genotyping was explored as an alternative to microsatellite markers. Fifty relevant SNPs were identified to predict the geographical origin of the samples, with an optimal F1-Score of 96% achieved with just 4 SNPs. The SNPs were ranked, with the most crucial one located on chromosome 9, position 48212140 (G/C), in an uncharacterized gene. An SNP genotyping tool is being developed to support law enforcement, providing accurate predictions about the origin of seized samples. The biological correlation of these SNPs remains open for future investigations. Beyond national impact, this work transcended borders, being presented at international scientific conferences and lectures for experts in Brazil. Notably, it was honored with the Outstanding Research Award in the forensic field during the 6th Forensic Meeting (6th EnqFor), further emphasizing the relevance and recognition of this research. In summary, this thesis not only addresses crucial issues related to Cannabis sativa but also offers practical and innovative solutions to combat drug trafficking, standing out for its international impact and recognition in the forensic arena