Structural annotation of equine protein‐coding genes determined by mRNA sequencing

Abstract

SummaryThe horse, like the majority of animal species, has a limited amount of species-specific expressed sequence data available in public databases. As a result, structural models for the majority of genes defined in the equine genome are predictions based on ab initio sequence analysis or the projection of gene structures from other mammalian species. The current study used Illumina-based sequencing of messenger RNA (RNA-seq) to help refine structural annotation of equine protein-coding genes and for a preliminary assessment of gene expression patterns. Sequencing of mRNA from eight equine tissues generated 293 758 105 sequence tags of 35 bases each, equalling 10.28 gbp of total sequence data. The tag alignments represent approximately 207· coverage of the equine mRNA transcriptome and confirmed transcriptional activity for roughly 90% of the protein-coding gene structures predicted by Ensembl and NCBI. Tag coverage was sufficient to refine the structural annotation for 11 356 of these predicted genes, while also identifying an additional 456 transcripts with exon/intron features that are not listed by either Ensembl or NCBI. Genomic locus data and intervals for the protein-coding genes predicted by the Ensembl and NCBI annotation pipelines were combined with 75 116 RNA-seq-derived transcriptional units to generate a consensus equine protein-coding gene set of 20 302 defined loci. Gene ontology annotation was used to compare the functional and structural categories of genes expressed in either a tissue-restricted pattern or broadly across all tissue samples.

Characterisation of the horse transcriptome from immunologically active tissues

“…With the difficulty in working with large animals, there is a lack of expressed sequence tag (EST) data, hence the current annotation of the protein coding regions of the horse genome is largely derived from extrapolation from the genes of other species (Coleman et al, 2010). This is beginning to be redressed with several recent papers outlining transcription profiles using digital gene analysis of a variety of horse tissues including muscle, leukocytes, cartilage, brain, reproductive tissue, embryos, sperm and blood (Capomaccio et al, 2013; Coleman et al, 2010; Das et al, 2013; Iqbal et al, 2014; McGivney et al, 2010; Park et al, 2012; Serteyn et al, 2010).…”

“…This is beginning to be redressed with several recent papers outlining transcription profiles using digital gene analysis of a variety of horse tissues including muscle, leukocytes, cartilage, brain, reproductive tissue, embryos, sperm and blood (Capomaccio et al, 2013; Coleman et al, 2010; Das et al, 2013; Iqbal et al, 2014; McGivney et al, 2010; Park et al, 2012; Serteyn et al, 2010). Capomaccio et al (2013) identified new putative non-coding sequences within intergenic and intronic regions whilst Das et al (2013) suggested additions to the structural annotation of four sperm genes.…”

RNA sequencing as a powerful tool in searching for genes influencing health and performance traits of horses

“…This modern method of analyzing global gene expression profiles was first used in horses in 2010 by Coleman et al With the use of the RNA-seq method, authors have analyzed th transcriptomes of several equine tissues (including cartilage, placenta, testis, cerebellum, embryonic tissue) and refined the structural annotation of more than 11,000 protein-coding genes annotated previously in the Ensembl database as predicted genes (Coleman et al 2010). Authors have also identified 456 new transcripts which were not listed in the Ensembl or NCBI databases.…”

“…Authors have also identified 456 new transcripts which were not listed in the Ensembl or NCBI databases. Moreover, Coleman et al (2010) reported tissue-specific expression profiles of thousands of genes and this type of research allowed for comprehensive functional and structural analysis of numerous genes in the horse genome. Further studies based on the results obtained from sequencing of the whole transcriptome (RNA-seq) allowed to identify 428 putative transcripts that have not been annotated in the Ensembl database and showing no homology to known protein-coding genes described in other species (Coleman et al 2013).…”

Genetic Susceptibility to Rhodococcus equi

“…To the authors' knowledge, the first report of RNA‐Seq in horses was an effort to characterize the transcriptome and tissue‐specific expression profiles from 8 equine tissues 94. A subsequent study focused on characterizing gene expression by RNA‐Seq in immunologically active tissues 95.…”