Background: 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, recent reports each utilizing RNA-seq to gauge X-linked gene expression relative to autosomal gene expression also arrived at diametrically opposed conclusions regarding X chromosome dosage compensation in mammals.

Results: 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.

Conclusions: Our analysis shows that the high number of paralogous gene families on the mammalian

X chromosome relative to autosomes contributes to the ambiguity in RXE calculations, RNA-seq analysis that

takes into account that single- and multi-copy genes are compensated differently supports the conclusion

that, in many somatic tissues, the mammalian X is up-regulated compared to the autosomes.