Abstract
Complete transcriptomic data at high resolution are available only for a few model organisms with medical importance. The gene structures of non-model organisms are mostly computationally predicted based on comparative genomics with other species. As a result, more than half of the horse gene models are known only by projection. Experimental data supporting these gene models are scarce. Moreover, most of the annotated equine genes are single-transcript genes. Utilizing RNA sequencing (RNA-seq) the experimental validation of predicted transcriptomes has become accessible at reasonable costs. To improve the horse genome annotation we performed RNA-seq on 561 samples of peripheral blood mononuclear cells (PBMCs) derived from 85 Warmblood horses. The mapped sequencing reads were used to build a new transcriptome assembly. The new assembly revealed many alternative isoforms associated to known genes or to those predicted by the Ensembl and/or Gnomon pipelines. We also identified 7,531 transcripts not associated with any horse gene annotated in public databases. Of these, 3,280 transcripts did not have a homologous match to any sequence deposited in the NCBI EST database suggesting horse specificity. The unknown transcripts were categorized as coding and noncoding based on predicted coding potential scores. Among them 230 transcripts had high coding potential score, at least 2 exons, and an open reading frame of at least 300 nt. We experimentally validated 9 new equine coding transcripts using RT-PCR and Sanger sequencing. Our results provide valuable detailed information on many transcripts yet to be annotated in the horse genome.
Highlights
Morphology studies have shown that equine and human lungs bear a striking resemblance [1,2]
There is limited information on the transcriptome profile of lung tissue and immune cells in horses. This limitation comes from the fact that there is only a small number of expressed sequence tags and cDNA data for horses deposited in public databases (37,756 entries in NCBI dbEST, release 130101)
We used samples stimulated with lipopolysaccharides (LPS of E. coli, Sigma–Aldrich) at a concentration of 250ng/ml; hay dust extract (HDE) [33,35] at three concentrations: 12, 9 or 6 μg/ml; or recombinant cyathostomin antigen (RCA) [36] at two concentrations: 4 or 1 μg/ml
Summary
Morphology studies have shown that equine and human lungs bear a striking resemblance [1,2]. In temperate climates 10% to 20% of stabled horses succumb to a condition called recurrent airway obstruction (RAO, known as heaves) characterized by stable dust-induced inflammation, bronchospasm and airway remodeling These characteristics are very similar to human asthma. The current gene models are derived based on a combination of ab initio methods, homology based studies, similarity and motif analysis programs This is currently rapidly changing with several groups publishing digital gene analyses from a variety of horse tissues, including muscle, leukocytes, cartilage, brain, reproductive tissue, embryos, sperm, and blood [6,7,8,9,10,11,12,13,14,15,16]. These studies have catalogued several non-coding genes in addition to protein coding gene isoforms, and structural annotations of existing genes
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