Computational protocol: Genetic variation and gene expression across multiple tissues and developmental stages in a non-human primate

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Protocol publication

[…] Real-time quantitative PCR was performed in two steps. First, reverse transcription (RT) was performed using the SuperScript® III First-Strand Synthesis System (Life Technologies) following the manufacturer’s protocol for priming with random hexamers. Custom primers and hydrolysis probes were designed for each lncRNA and three candidate reference genes (Hypoxanthine phosphoribosyltransferase 1, HPRT1; Glyceraldehyde 3-phosphate dehydrogenase, GAPDH; and Beta-2-Microglobulin, B2M) using the Custom TaqMan® Assays Design Tool (Applied Biosystems, ). Expression analyses were conducted on the LightCycler™ 480 platform (Roche) using the iTaq® Universal Probes Supermix (Bio-Rad). All qPCR reactions were carried out in triplicate; reactions containing water instead of cDNA were included as negative controls. cDNA samples were diluted 1:5 with water, and a five-point standard curve of four-fold dilutions was prepared for each gene using pooled cDNA as the template. Stability of each candidate reference gene was evaluated using the NormFinder software (v5) in R. Quantification was performed using the relative standard curve method, with the geometric mean of the most stably expressed reference genes (GAPDH and HPRT1) used as an endogenous control for normalization of the interpolated lncRNA quantities. Finally, relative expression levels were generated by dividing the normalized lncRNA quantities by the corresponding quantity in one experimental sample which served as a calibrator. For additional experimental details and complete primer and probe sequence information see . [...] Genotypes were generated through WGS, as described previously. Genotypes from 721 VRC vervets that passed QC procedures can be queried via the EVA at EBI. Two genotype data sets were used: (1) The Association Mapping Set consists of 497,163 SNPs on the 29 vervet autosomes. This set has, on average, 198 SNPs per Mb of vervet sequence, with a maximal gap of 5 Kb between adjacent SNPs. (2) The Linkage Mapping Set consists of 147,967 SNPs on the 29 vervet autosomes. This set has, on average, 58.2 SNPs per Mb of vervet sequence, with an average gap of 17.5 Kb between adjacent SNPs.The software package Loki, which implements Markov Chain Monte Carlo methods, was used to estimate multipoint identical by decent (MIBD) allele-sharing among all vervet family members from the genotype data. As long stretches of IBD were evident among these closely related animals, density 9,752 SNPs of the 148K set were sufficient to evaluate MIBD at 1cM intervals. The correspondence between physical and genetic positions of vervet SNPs was established by interpolation using 360 markers from the vervet STR linkage map, for which physical and genetic position was known. […]

Pipeline specifications

Software tools NormFinder, Loki
Applications WGS analysis, qPCR
Organisms Chlorocebus aethiops, Homo sapiens