Determination of Dosage Compensation of the Mammalian X Chromosome by RNA-Seq is Dependent on Analytical Approach

An enduring question surrounding sex chromosome evolution is whether effective hemizygosity in the heterogametic sex leads inevitably to dosage compensation of sex-linked genes, and whether this compensation has been observed in a variety of organisms. Incongruence in the conclusions reached in some recent reports has been attributed to different high-throughput approaches to transcriptome analysis. However, two recent reports that both utilize RNA-seq to gauge X-linked gene expression relative to autosomal gene expression arrive at diametrically opposed conclusions regarding X chromosome dosage compensation in mammals. Here we analyze RNA-seq data from X-monosomic female human and mouse tissues, which are uncomplicated by genes that escape X-inactivation, as well as published RNA-seq data to describe relative X expression (RXE). We find that the determination of RXE is highly dependent upon a variety of computational, statistical and biological assumptions underlying RNA-seq analysis. Parameters implemented in short-read mapping programs, choice of reference genome annotation, expression data distribution, tissue source for RNA and RNA-seq library construction method have profound effects on comparing expression levels across chromosomes. Our analysis shows that the mammalian X chromosome is highly enriched for paralogous gene families compared to autosomes, and that single- and multi-copy genes are compensated differently. RNA-seq analysis that takes this into account supports the conclusion that, in many somatic tissues, the mammalian X is up-regulated compared to the autosomes.