Project description:Purpose:The goal of this study was to evalute gene expression patterns of equine chorioallantoic membrane during different stages of the pregnancy Method: mRNA profile of equine chorioallantoic membrane (CAM) from 45days, 4months, 6months and 10months (4 samples for each time points) generated by RNA-sequencing,using a Illumina HiSeq 4000 ( HiSeq 4000 sequencing kit version 1). The sequence reads were trimmed for adapters and quality using TrimGalore Version 0.4.4,and then mapped to EquCab2.0 using STAR-2.5.2b. Final quantification at the gen level was performed by analyzing the BAM files in cufflinks using the Equus_caballus_ENSEMBL_88 gtf file as Guide.
Project description:Through this comparative proteomic study of equine CD4+ cells 376 membrane proteins could be identified. Several proteins showed changed abundance in horses affected by a spontaneous autoimmune disease (equine recurrent uveitis, ERU). Findings provide novel knowledge about changes in the CD4+ immune cell membrane proteome in a spontaneously and naturally occurring autoimmune disease in horses. The data is highly relevant for veterinary medicine and has proven translational quality for autoimmune uveitis in man.
Project description:Horse-specific genes are not readily identified from available equine EST/cDNA resources due to relatively limited coverage. In addition, equine gene sets predicted in silico by Ensembl and NCBI will not identify horse specific genes since they rely on homology-based projection of gene structure annotation from other species. In this study, RNA-seq of 8 equine RNA samples representing 6 distinct tissues was performed and used to improve and refine equine gene structure annotation. The samples and RNA were collected as part of the related study E-GEOD-21925 and are described in Coleman et al 2010. Anim Genet 41 Suppl 2: 121-30 (PMID: 21070285). The RNA from these samples was re-sequenced in this experiment. The tissues were i). the articular cartilage and synovial membrane samples from a 3-year-old male pony. The left carpal joints received four LPS injections (0.5 ng) over 8 days, while the right carpal joints received control injections of PBS. ii) A cerebellum sample was collected from a 2-year-old female thoroughbred. iii) A testis sample from a 4-year-old thoroughbred. iv) A placental villous sample collected immediately post-partum from a full-term female thoroughbred foal. v) A whole embryo sample was obtained from a 34-day-old male thoroughbred conceptus. The embryo, cerebellum, testis and placental samples were of apparent normal gross morphology.
Project description:The horse, like a majority of animal species, has a limited amount of species-specific expressed sequence data available in public databases. As a result, structural models for a 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 thirty five-base sequence tags, equaling 10.28 giga-basepairs of total sequence data. The tag alignments represent approximately 208X 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 define structural annotation for 11,356 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. Examination of 8 equine RNA samples representing 6 distinct tissues
Project description:Sequencing of equine mRNA (RNA-seq) identified 428 putative transcripts which do not map to any previously annotated or predicted horse genes. Most of these encode the equine homologs of known protein-coding genes described in other species, yet the potential exists to identify novel and perhaps equine-specific gene structures. A set of 36 transcripts were prioritized for further study by filtering for levels of expression (depth of RNA-seq read coverage), distance from annotated features in the equine genome, the number of putative exons, and patterns of gene expression between tissues. From these, four were selected for further investigation based on predicted open reading frames of greater than or equal to 50 amino acids and lack of detectable homology to known genes across species. Sanger sequencing of RT-PCR amplicons from additional equine samples confirmed expression and structural annotation of each transcript. Functional predictions were made by conserved domain searches. A single transcript, expressed in the cerebellum, contains a putative kruppel-associated box (KRAB) domain, suggesting a potential function associated with zinc finger proteins and transcriptional regulation. Overall levels of conserved synteny and sequence conservation across a 1MB region surrounding each transcript were approximately 73% compared to the human, canine, and bovine genomes; however, the four loci display some areas of low conservation and sequence inversion in regions that immediately flank these previously unannotated equine transcripts. Taken together, the evidence suggests that these four transcripts are likely to be equine-specific.
Project description:The horse, like a majority of animal species, has a limited amount of species-specific expressed sequence data available in public databases. As a result, structural models for a 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 thirty five-base sequence tags, equaling 10.28 giga-basepairs of total sequence data. The tag alignments represent approximately 208X 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 define structural annotation for 11,356 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.
Project description:Standardized muscular biopsies of the dorsal compartment of the gluteus medius muscle were performed in 7 horses suffering from equine polysaccharide storage myopathy (PSSM) and 6 sound Norman Cob horses . Gene expression analysis was performed using an equine oligonucleotide microarray which included 384 equine gene probes of the nuclear genome and all the mitochondrial genes. All the samples of PSSM muscles were hybridized against the reference control muscles. This reference was made by pooling together all the mRNA extracted after in vitro transcription from the 6 control muscles of the sound horses. Briefly, the hybridization protocol was adapted from Le Brigand et al. (2006). An open-access long oligonucleotide microarray resource for analysis of the human and mouse transcriptomes. Nucleic Acids Res. 2006 Jul 19;34(12).
Project description:A tissue survey of gene expression was conducted using microarray-based transcriptional profiling to compare equine articular cartilage to 10 other normal adult horse tissues. The ten comparative tissues were bladder, cerebellum, kidney, liver, lung, lymph node, muscle, placental villous, spleen, and testis. Messenger RNA transcriptome comparisons were conducted between equine articular cartilage and ten other body tissues using a 9413 element equine-specific cDNA microarray and a two-color dye-swap experimental design. After scanning, the median intensities adjusted for background were entire chip Lowess-normalized for each individual slide. Quantile regression was used to estimate the conditional quantile of the M and A log ratios given the observed average log intensity. Briefly, a nonparametric approach was used to reveal the relationship between percentiles of M and A, where M is log2 (R/G) and A is 0.5 log2 (RG) with R representing expression in articular cartilage and G representing expression in the comparative tissue. The quantile regression was fit using a B-spline with 5 fixed nodes. The 1st, 5th, 10th, 20th, 50th, 80th, 90th, 95th, and 99th conditional quantiles were estimated. For each observed gene intensity in a given tissue comparison, the normal quantile was used as the cartilage-specificity in place of the corresponding estimated regression quantile.