Computational protocol: Genetic Analysis of a Rat Model of Aerobic Capacity and Metabolic Fitness

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Protocol publication

[…] DNA from 22-25 breeders from both lines in three non-adjacent generations (G5, G14, and G26, n=142) was extracted from frozen liver tissue, and genotyped across 10,846 SNP loci using the Affymetrix Rat Mapping 10K GeneChip. Attempts to extract DNA from generations earlier than G5 revealed that many samples in G0 and G4 were degraded. We therefore chose G5 as the earliest generation in our analysis due to its assured DNA quality. In assessing the quality of SNP markers we removed 28 duplicate SNPs, 496 SNPs with genotype missing rate >10%, and 137 SNPs with Hardy-Weinberg Equilibrium test p < 0.001. These steps led to 10,185 SNPs that formed the "Panel-1" markers. As some analyses require a reduced set of SNPs without rare variants and without strong linkage disequilibrium, we removed from Panel-1 an addition set of 7,284 SNPs selected by trimming SNP pairs in linkage disequilibrium with r2 value >0.05 (in windows of 10 SNPs, sliding by 2 SNPs each time), and 572 SNPs with minor allele frequency (MAF) <5%. After these steps, 2,518 SNPs remained and formed the "Panel-2" markers. The Panel-2 markers were used in calculations of IBD, AMOVA, and genome-wide average heterozygosity. Pairwise Identity-by-State (IBS) matrix was estimated in PLINK [] using the -genome command and Panel-2 markers. Multidimensional scaling analysis of the IBS matrix was performed in R []. Analysis of molecular variance (AMOVA), as implemented in the program Arlequin, was used to calculate the within- and among-group differentiation []. We assessed the accuracy of recorded sex for each genotyped animal by calculating the average heterozygosity of the X chromosome SNPs. Male and female animals are confirmed by non-overlapping distributions of ChrX heterozygosity values. We confirmed known sibling pairs among the genotyped animals by plotting pairwise Z0 vs Z1 values in R. Z0 and Z1 values were determined in PLINK using the -genome command. [...] Pairwise measurements of LD (r2) were calculated for marker pairs within 5 Mb on Chromosome 1 using Haploview []. Chromosome 1 was chosen as a representaive autosome. To show the relationship between r2 and inter-marker distance, we calculated average r2 values for groups of marker pairs falling in discrete bins of inter-marker distance, in 500Kb increments, and plotted the values for G5, G14, and G26 in both HCR and LCR (as shown in ). […]

Pipeline specifications

Software tools PLINK, Arlequin, Haploview
Applications Population genetic analysis, GWAS
Organisms Rattus norvegicus, Homo sapiens