Abstract The advent of next-generation sequencing allows simultaneous processing of several genomic regions/individuals, increasing the availability and accuracy of whole-genome data. However, these new approaches may present some errors and bias due to alignment, genotype calling, and imputation methods. Despite these flaws, data obtained by next-generation sequencing can be valuable for population and evolutionary studies of specific genes, such as genes related to how pigmentation evolved among populations, one of the main topics in human evolutionary biology. Melanocortin-1 receptor (MC1R) is one of the most studied genes involved in pigmentation variation. As MC1R has already been suggested to affect melanogenesis and increase risk of developing melanoma, it constitutes one of the best models to understand how natural selection acts on pigmentation. Here we employed a locally developed pipeline to obtain genotype and haplotype data for MC1R from the raw sequencing data provided by the 1000 Genomes FTP site. We also compared such genotype data to Phase 3 VCF to evaluate its quality and discover any polymorphic sites that may have been overlooked. In conclusion, either the VCF file or one of the presently described pipelines could be used to obtain reliable and accurate genotype calling from the 1000 Genomes Phase 3 data.

O teste padrão para identificar o potencial cancerígeno de compostos químicos é o estudo de longa duração em roedores, não realizado no Brasil. Nosso laboratório estabeleceu um teste alternativo de média duração (ensaio DMBDD), baseado no paradigma iniciação-promoção da carcinogênese química, adotado pelo Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (Ibama) como fonte oficial de evidências de carcinogenicidade. Nosso objetivo foi avaliar alterações nos éxons 5 a 8 do gene supressor tumoral TP53 e éxons 1 e 2 dos oncogenes H-RAS e K-RAS em lesões hepáticas neoplásicas e pré-neoplásicas observadas no ensaio DMBDD. A identificação dessas eventuais alterações moleculares permitirá o reconhecimento de um possível padrão de alteração em genes críticos, bem como poderão sugerir mecanismos de ação das substâncias testadas no protocolo DMBDD. Sessenta ratos wistar machos foram separados em três grupos: o grupo I não foi tratado quimicamente; o grupo II recebeu os cinco agentes iniciadores da carcinogênese (DMBDD); o grupo III recebeu a iniciação DMBDD seguida de promoção por Fenobarbital. Amostras de DNA (obtidas após avaliação histopatológica de fígado fixado em formalina e incluído em parafina) foram submetidas à técnica da PCR-SSCP (single strand conformation polymorphism). Não foram detectadas mudanças no padrão de bandeamento da SSCP nos diferentes grupos experimentais em nenhum dos éxons estudados. Os resultados sugerem que, em fígado de ratos Wistar machos, mutações nos éxons 5 a 8 do gene TP53 e éxons 1 e 2 dos genes H-RAS e K-RAS não se incluem dentre as alterações moleculares precoces do processo de carcinogênese hepática induzido pelo protocolo DMBDD.

The standard protocol to evaluate the carcinogenic potential of chemicals is the long-term bioassay in rodents, not performed in developing countries due to its high cost and complex operational procedures. Our laboratory has established an alternative medium-term bioassay in Wistar rats, also called DMBDD assay, based on the paradigm initiation/promotion of chemical carcinogenesis. This method was accepted by the Brazilian Environment Agency (IBAMA) as an official source of evidence of carcinogenicity. The aim of this study was to evaluate alterations in exons 5 to 8 of the tumor suppressor gene TP53 and exons 1 and 2 of oncogenes K-RAS and H-RAS in neoplastic and preneoplastic hepatic lesions observed in DMBDD assay. The characterization of these alterations may contribute to the recognition of patterns of damage in critical genes, as well as to suggest mechanisms of action of the compounds tested in the protocol. Sixty male Wistar rats were separated into 3 groups: the first was treated with no chemicals; the second received five initiating agents and the third received initiation followed by phenobarbital. Liver DNA samples (obtained from formalin-fixed and paraffin-embedded tissues after histological analysis) were evaluated by the non-isotopic PCR-SSCP technique. No changes in any analyzed exons were detected by the PCR-SSCP banding pattern in all experimental groups. This result suggests that liver mutations in exons 5 to 8 of TP53 and exons 1 and 2 of H-RAS and K-RAS are not among the early molecular alterations occurring in the hepatic carcinogenesis process induced by the DMBDD protocol in male Wistar rats.