The authors provide Gene Ontology analyses to demonstrate how the allele-specific expression differences may impact on the interpretation of differential gene expression caused by a mutation. Using a variety of mutant and wild-type alleles with sophisticated analysis of RNA-seq data in zebrafish embryos they demonstrate over-representation of expression changes of genes in linkage with the mutant allele on the same chromosome. White and colleagues tested the hypothesis that genes in linkage with a mutant allele might show allele-specific expression differences and thus potentially confound the interpretation of mutant effects. Zebrafish strains are typically considerably polymorphic. These observations have implications for the interpretation of RNA-seq experiments involving outbred animals and non-inbred model organisms. This could lead to pathways being erroneously implicated or overlooked due to the noise of spurious differentially expressed genes on the same chromosome as the mutation. Typically, the genomic location of a differentially expressed gene is not considered when interpreting its importance with respect to the phenotype. When analysing mutant transcriptomes, this means that the differential expression of genes on the same chromosome as a mutation of interest may not be caused by that mutation. Using an incross of haplotype-resolved wild-type fish, we found evidence of widespread allele-specific expression, which appears as differential expression when comparing embryos homozygous for a region of the genome to their siblings. Here we show that in zebrafish, differentially expressed genes are often over-represented on the same chromosome as the mutation due to different levels of expression of alleles from different genetic backgrounds.

Genes that are differentially expressed between the groups are assumed to reveal insights into the pathways affected by the mutation.

Resultant embryos are grouped by genotype to compare homozygous mutant embryos to heterozygous and wild-type siblings. Typically, animals heterozygous for a mutation are crossed to produce offspring with different genotypes. In model organisms, RNA-sequencing (RNA-seq) is frequently used to assess the effect of genetic mutations on cellular and developmental processes.

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