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

Abstract 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. Author summary The human X and Y chromosomes share an evolutionary origin and sequence homology, including regions of 100% identity; this sequence homology can result in reads misaligning between the sex chromosomes, X and Y. We hypothesized that misalignment of reads on the sex chromosomes would confound estimates of transcript abundance if the sex chromosome complement of the sample is not accounted for during the alignment step. For example, because of shared sequence similarity, X-linked reads could misalign to the Y chromosome. This is expected to result in reduced expression for regions between X and Y that share high levels of homology. For this reason, we tested the effect of using a default reference genome versus a reference genome informed by the sex chromosome complement of the sample on estimates of transcript abundance in human RNA-Seq samples from the brain cortex and whole blood of three genetic female (46, XX) and three genetic male (46, XY) samples. We found that using a reference genome with the sex chromosome complement of the sample resulted in higher measurements of X-linked gene transcription for both male and female samples and more differentially expressed genes on the X and Y chromosomes. We recommend future studies requiring aligning reads to a reference genome should consider the sex chromosome complement of their samples prior to running default pipelines.