Reference genome and transcriptome informed by the sex chromosome complement of the sample increase ability to detect sex differences in gene expression from RNA-Seq data
Open Access
- 21 July 2020
- journal article
- research article
- Published by Springer Science and Business Media LLC in Biology of Sex Differences
- Vol. 11 (1), 1-18
- https://doi.org/10.1186/s13293-020-00312-9
Abstract
Human 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. 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 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. 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 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.Keywords
Funding Information
- National Institute of General Medical Sciences (R35GM124827)
- Biodesign Institute, Arizona State University (Startup)
- School of Life Sciences, Arizona State University (Startup)
This publication has 68 references indexed in Scilit:
- The Cancer Genome Atlas Pan-Cancer analysis projectNature Genetics, 2013
- STAR: ultrafast universal RNA-seq alignerBioinformatics, 2012
- GENCODE: The reference human genome annotation for The ENCODE ProjectGenome Research, 2012
- BamTools: a C++ API and toolkit for analyzing and managing BAM filesBioinformatics, 2011
- The Sequence Read ArchiveNucleic Acids Research, 2010
- edgeR: a Bioconductor package for differential expression analysis of digital gene expression dataBioinformatics, 2009
- The Sequence Alignment/Map format and SAMtoolsBioinformatics, 2009
- What does the “four core genotypes” mouse model tell us about sex differences in the brain and other tissues?Frontiers in Neuroendocrinology, 2009
- X-inactivation profile reveals extensive variability in X-linked gene expression in femalesNature, 2005
- The male-specific region of the human Y chromosome is a mosaic of discrete sequence classesNature, 2003