Computational protocol: Faster-X Evolution of Gene Expression in Drosophila

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Protocol publication

[…] We obtained microarray measurements of expression from whole fly or head from previously published results –. We calculated the expression level of each gene by first taking the median signal across all probes for each gene within each replicate, and then calculating the median for each gene across all replicates. As an alternative approach, we used expression levels estimated in the LIMMA package of Bioconductor , as described previously .We tested for significant differences in expression between males and females (i.e., sex-biased expression) using moderated t-tests implemented in the LIMMA package with the empirical Bayes function to pool sample variances toward a common value , as described previously . We corrected for multiple tests using a FDR of 0.05 when only sex-biased expression in D. melanogaster was considered, and with a FDR of 0.20 when sex-biased expression in all species was considered. Genes with significantly higher expression in males were classified as having male-biased expression, and those with higher expression in females as female-biased.RNA-seq data collected from whole D. melanogaster, D. pseudoobscura, and D. mojavensis males and female or D. melanogaster, D. pseudoobscura, D. willistoni, and D. mojavensis heads were obtained from previously published results . Reads longer than 36 bases (bp) were trimmed to 36 bp so that all reads were the same length, and reads were then mapped to the transcriptome using BWA . Any read mapping to multiple locations in the genome was discarded, and genes with fewer than 50 mapped reads were excluded from the subsequent analysis. The expression level of each gene was estimated as the number of unique reads mapping to the gene standardized by the total number of mapped reads and the transcript length.We extracted only those genes present as 1∶1 orthologs on the same chromosome arm in all species under consideration, and we quantile normalized the expression levels so that they are identically distributed across all species. We next calculated SpearmanÕs between all pairwise comparisons of expression levels from the species under consideration. This was repeated for each chromosome arm. Confidence intervals (CIs) of were estimated by bootstrapping the data 1000 times in the R statistical computing environment . We also calculated correlations of expression between sexes within each species.Microarray measurements of expression were obtained for 14 adult D. melanogaster tissues from FlyAtlas , and the expression breadth was determined for each gene as described previously . Briefly, we calculated , a metric that ranges between 0 (for broadly expressed genes) to 1 (for narrowly expressed genes). Genes were said to be narrowly expressed in a tissue if , and genes with were classified as broadly expressed. […]

Pipeline specifications

Software tools limma, BWA
Databases FlyAtlas
Application RNA-seq analysis
Organisms Drosophila melanogaster, Homo sapiens