Abstract

BackgroundHuman X and Y chromosomes share an evolutionary origin and, as a consequence, sequence similarity. We investigated whether the sequence homology between the X and Y chromosomes affects the alignment of RNA-Seq reads and estimates of differential expression. We tested the effects of using reference genomes and reference transcriptomes informed by the sex chromosome complement of the sample’s genome on the measurements of RNA-Seq abundance and sex differences in expression.ResultsThe 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 the transcript abundance in the whole blood, brain cortex, breast, liver, and thyroid tissues from 20 genetic female (46, XX) and 20 genetic male (46, XY) samples. 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, repeated using two different read aligners, HISAT and STAR. 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. We additionally created sex chromosome complement informed transcriptome references for use in pseudo-alignment using Salmon. Transcript abundance was quantified twice for each sample: once to the default target transcripts and then independently to target transcripts informed by the sex chromosome complement of the sample.ConclusionsWe show that regardless of the choice of the read aligner, using an alignment protocol informed by the sex chromosome complement of the sample results in higher expression estimates on the pseudoautosomal regions of the X chromosome in both genetic male and genetic female samples, as well as an increased number of unique genes being called as differentially expressed between the sexes. We additionally show that using a pseudo-alignment approach informed on the sex chromosome complement of the sample eliminates Y-linked expression in female XX samples.

Highlights

  • Human X and Y chromosomes share an evolutionary origin and, as a consequence, sequence similarity

  • We show that regardless of the choice of the read aligner, using an alignment protocol informed by the sex chromosome complement of the sample results in higher expression estimates on the pseudoautosomal regions of the X chromosome in both genetic male and genetic female samples, as well as an increased number of unique genes being called as differentially expressed between the sexes

  • We show that using a pseudo-alignment approach informed on the sex chromosome complement of the sample eliminates Y-linked expression in female XX samples

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Summary

Introduction

Human X and Y chromosomes share an evolutionary origin and, as a consequence, sequence similarity. Accounting for the sex chromosome complement of the sample in quantifying gene expression has been limited due to shared sequence homology between the sex chromosomes, X and Y, that can confound gene expression estimates. The X and Y chromosomes share an evolutionary origin: mammalian X and Y chromosomes originated from a pair of indistinguishable autosomes ~ 180–210 million years ago that acquired the sex-determining genes [9,10,11]. Other regions of high sequence similarity between X and Y include the X-transposed region (XTR) with 98.78% homology [15] (Fig. 1a). The evolution of the X and Y chromosomes has resulted in a pair of chromosomes that are diverged, but still share some regions of high sequence similarity

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