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

Abstract

Background: 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. 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 hardmasked 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

Gene-rich UV sex chromosomes harbor conserved regulators of sexual development

“…Filtered reads were mapped using HISAT2 (v2.1.0) ( 84 ) to the C. purpureus R40 genome (autosomes and V sex chromosome) concatenated with the GG1 U sex chromosome. We hard masked the U chromosome for males, and the V for females, using Bedtools (v2.27.1) ( 76 ) maskfasta ( 85 ). Genes greater than 300 bp were assembled using StringTie (v1.3.3) ( 86 ), gene counts were extracted using StringTie’s prepDE.py script ( http://ccb.jhu.edu/software/stringtie/index.shtml?t=manual#deseq ), and gene IDs renamed (using mstrg_prep.pl, https://gist.github.com/gpertea/b83f1b32435e166afa92a2d388527f4b ).…”

Genome-Wide Sex and Gender Differences in Cancer

“…This results in sequencing data that contains poor quality mapping of similar regions between the sex chromosomes, and spurious reads mapped to the Y chromosome in samples from XX genomes. A recent study showed that accounting for these artifacts in sequence mapping protocols can improve variant calling (153) and detection of sex differential gene expression (154). The XYalign is a sex-informed sequence alignment method that first identifies whether the sequencing reads derives from an XX or XY genome based on read balance, and then align the sequencing reads to a sex-appropriate reference genome (153).…”

Shared Gene Expression and Immune Pathway Changes Associated with Progression from Nevi to Melanoma

“…Short sequencing reads from the X and Y chromosomes may mismap due to the high degree of sequence homology. To account for this mismapping, female samples were mapped to the GENCODE GRCh38 reference genome with the Y-chromosome hard-masked, and male samples were mapped to the GRCH38 reference genome with the Y-chromosomal pseudo-autosomal regions hard-masked [ 19 , 20 ]. Raw read counts were summed across all transcripts of the same gene to give a gene-level expression, and genes were only included if they had a known gene-level annotation.…”