Computational protocol: Association of Genetic Variants with Self-Assessed Color Categories in Brazilians

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Protocol publication

[…] The chosen SNPs were genotyped using the real-time PCR TaqMan assay utilizing two differentially fluorescently labeled probes that permitted the detection of both alleles in a single reaction (Applied Biosystems INC, Foster City, CA, USA). The PCR primers and Taqman probes had been previously developed at Applied Biosystems and are listed in Supplementary Table S1 for each SNP studied. Assays were performed on an ABI 7900 HT Fast PCR Real Time System (Applied Biosystems, Foster City, CA), and the genotype assignments were conducted using the TaqMan™ Genotyper Software, in a 384-well format and using manufacturer's instructions.The classification accuracy of each TaqMan assay was validated by cycle sequencing (forward and reverse) of PCR fragments containing the polymorphisms studied using the DYEnamicTM ET Dye Terminator Kit, (GE Healthcare) standard procedure and a MegaBACE™ 1000 sequencer (GE Healthcare). After the run in the MegaBACE sequencer, the electrofluorograms were visualized using Sequencher™ v 4.1.4 (Gene Codes Corporation, Ann Arbor, USA). [...] The regression analysis was performed to study the association of each polymorphism with self-classified skin color categories, the phenotype was numerically fixed as dependent variable, the genotypic data were fixed as independent variables and the ancestry values were fixed as covariates. Initially we made a numerical linear regression using Golden Helix SNP and Variation Suite software (SVS Version 7.2.2, (Golden Helix Inc., Bozeman, MT, USA). In addition, a partial regression model (from the same package) called Reduced x Full model was used with a type I error α of 5%, considering the Bonferroni correction. We introduced individual ancestry as a covariate in this last model in order to control spurious associations that may be the result of differences in the ancestral proportions (admixture). When the results (P values) of the two models were compared to determine which loci remain associated with the phenotype even after the correction for covariates (ancestry). For all SNPs numerical regression analyses were performed considering three genetic models: additive, dominant and recessive. The statistical significance was defined as p<0.05. P values were corrected by the Bonferroni adjustment for multiple comparisons tests. […]

Pipeline specifications

Software tools genotypeR, Sequencher, SVS, ADMIXTURE
Applications Population genetic analysis, GBS analysis
Organisms Homo sapiens