Background: Human X and Y chromosomes share an evolutionary origin and, as a consequence, sequence similarity. We investigated whether sequence homology between the X and Y chromosomes affects alignment of RNA-Seq reads, and estimates of differential expression. We tested the effects of using reference genomes informed by the sex chromosome complement of the sample's genome on measurements of RNA-Seq abundance and sex-differences in expression. Results: The default genome includes the entire human reference genome (GRCh38), including the entire sequence of the X and Y chromosomes. We created two sex chromosome complement informed reference genomes. One sex chromosome complement informed reference genome was used for samples that lacked a Y chromosome; for this reference genome version, we hard-masked the entire Y chromosome. For the other sex chromosome complement informed reference genome, to be used for samples with a Y chromosome, we hard-masked only the pseudoautosomal regions of the Y chromosome, because these regions are duplicated identically in the reference genome on the X chromosome. We analyzed transcript abundance in the brain cortex, and whole blood from three genetic female (46, XX) and three genetic male (46, XY) samples, using both HISAT and STAR read aligners. Each sample was aligned twice; once to the default reference genome and then independently aligned to a reference genome informed by the sex chromosome complement of the sample. We then quantified sex-differences in gene expression using featureCounts to get the raw count estimates followed by Limma/Voom for normalization and differential expression. Conclusions: We show that regardless of the choice of read aligner, using an alignment protocol informed by the sex chromosome complement of the sample results in higher expression estimates on the X chromosome in both genetic male and genetic female samples and an increased number of unique genes being called as differentially expressed between the sexes.

Aligning RNA-Seq reads to a sex chromosome complement informed reference genome increases ability to detect sex differences in gene expression

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