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Generation of a de novo transcriptome from equine lamellar tissue.

ABSTRACT: Laminitis, the structural failure of interdigitated tissue that suspends the distal skeleton within the hoof capsule, is a devastating disease that is the second leading cause of both lameness and euthanasia in the horse. Current transcriptomic research focuses on the expression of known genes. However, as this tissue is quite unique and equine gene annotation is largely derived from computational predictions, there are likely yet uncharacterized transcripts that may be involved in the etiology of laminitis. In order to create a novel annotation resource, we performed whole transcriptome sequencing of sagittal lamellar sections from one control and two laminitis affected horses.Whole transcriptome sequencing of the three samples resulted in 113 million reads. Overall, 88 % of the reads mapped to the equCab2 reference genome, allowing for the identification of 119,430 SNPs. The de novo assembly generated around 75,000 transcripts, of which 36,000 corresponded to known annotations. Annotated transcript models are hosted in a public data repository and thus can be easily accessed or loaded into genome browsers. RT-PCR of 12 selected assemblies confirmed structure and expression in lamellar tissue.Transcriptome sequencing represents a powerful tool to expand on equine annotation and identify novel targets for further laminitis research.

SUBMITTER: Holl HM

PROVIDER: S-EPMC4592545 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Generation of a de novo transcriptome from equine lamellar tissue.

Holl Heather M HM Gao Shan S Fei Zhangjun Z Andrews Caroline C Brooks Samantha A SA

BMC genomics 20151003

<h4>Background</h4>Laminitis, the structural failure of interdigitated tissue that suspends the distal skeleton within the hoof capsule, is a devastating disease that is the second leading cause of both lameness and euthanasia in the horse. Current transcriptomic research focuses on the expression of known genes. However, as this tissue is quite unique and equine gene annotation is largely derived from computational predictions, there are likely yet uncharacterized transcripts that may be involv ...[more]

PMID: 26432030

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Project description:Endocrinopathic laminitis is pathologically similar to the multi-organ dysfunction and peripheral neuropathy found in human patients with metabolic syndrome. Similarly, endocrinopathic laminitis has been shown to partially result from vascular dysfunction. However, despite extensive research, the pathogenesis of this disease is not well elucidated and laminitis remains without an effective treatment. Here, we sought to identify novel proteins and pathways underlying the development of equine endocrinopathic laminitis. Healthy Standardbred horses (n = 4/group) were either given an electrolyte infusion, or a 48-h euglycemic-hyperinsulinemic clamp. Cardiac and lamellar tissues were analyzed by mass spectrometry (FDR = 0.05). All hyperinsulinemic horses developed laminitis despite being previously healthy. We identified 514 and 709 unique proteins in the cardiac and lamellar proteomes, respectively. In the lamellar tissue, we identified 14 proteins for which their abundance was significantly increased and 13 proteins which were significantly decreased in the hyperinsulinemic group as compared to controls. These results were confirmed via real-time reverse-transcriptase PCR. A STRING analysis of protein-protein interactions revealed that these increased proteins were primarily involved in coagulation and complement cascades, platelet activity, and ribosomal function, while decreased proteins were involved in focal adhesions, spliceosomes, and cell-cell matrices. Novel significant differentially expressed proteins associated with hyperinsulinemia-induced laminitis include talin-1, vinculin, cadherin-13, fibrinogen, alpha-2-macroglobulin, and heat shock protein 90. In contrast, no proteins were found to be significantly differentially expressed in the heart of hyperinsulinemic horses compared to controls. Together, these data indicate that while hyperinsulinemia induced, in part, microvascular damage, complement activation, and ribosomal dysfunction in the lamellae, a similar effect was not seen in the heart. In brief, this proteomic investigation of a unique equine model of hyperinsulinemia identified novel proteins and signaling pathways, which may lead to the discovery of molecular biomarkers and/or therapeutic targets for endocrinopathic laminitis.

Project description:BACKGROUND: The tropical green-lipped mussel Perna viridis is a common biomonitor throughout the Indo-Pacific region that is used for environmental monitoring and ecotoxicological investigations. However, there is limited molecular data available regarding this species. We sought to establish a global transcriptome database from the tissues of adductor muscle, gills and the hepatopancreas of P. viridis in an effort to advance our understanding of the molecular aspects involved during specific toxicity responses in this sentinel species. RESULTS: Illumina sequencing results yielded 544,272,542 high-quality filtered reads. After de novo assembly using Trinity, 233,257 contigs were generated with an average length of 1,264 bp and an N50 length of 2,868 bp; 192,879 assembled transcripts and 150,111 assembled unigenes were obtained after clustering. A total of 93,668 assembled transcripts (66,692 assembled genes) with putative functions for protein domains were predicted based on InterProScan analysis. Based on similarity searches, 44,713 assembled transcripts and 25,319 assembled unigenes were annotated with at least one BLAST hit. A total of 21,262 assembled transcripts (11,947 assembled genes) were annotated with at least one well-defined Gene Ontology (GO) and 5,131 assembled transcripts (3,181 assembled unigenes) were assigned to 329 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The quantity of assembled unigenes and transcripts obtained from male and female mussels were similar but varied among the three studied tissues, with the highest numbers recorded in the gills, followed by the hepatopancreas, and then the adductor muscle. Multivariate analyses revealed strong tissue-specific patterns among the three different tissues, but not between sexes in terms of expression profiles for annotated genes in various GO terms, and genes associated with stress responses and degradation of xenobiotics. The expression profiles of certain selected genes in each tissue type were further validated using real-time quantitative polymerase chain reaction assays and a similar tissue-specific trend was seen. CONCLUSIONS: The extensive sequence data generated from this study will provide a valuable molecular resource for facilitating environmental studies with P. viridis, and highlight the importance of tissue-specific approaches in the future.

Project description:BackgroundBarnyardgrass (Echinochloa crus-galli) is an important weed that is a menace to rice cultivation and production. Rapid evolution of herbicide resistance in this weed makes it one of the most difficult to manage using herbicides. Since genome-wide sequence data for barnyardgrass is limited, we sequenced the transcriptomes of susceptible and resistant barnyardgrass biotypes using the 454 GS-FLX platform.Results454 pyrosequencing generated 371,281 raw reads with an average length of 341.8 bp, which made a total length of 126.89 Mb (SRX160526). De novo assembly produced 10,142 contigs (∼5.92 Mb) with an average length of 583 bp and 68,940 singletons (∼22.13 Mb) with an average length of 321 bp. About 244,653 GO term assignments to the biological process, cellular component and molecular function categories were obtained. A total of 6,092 contigs and singletons with 2,515 enzyme commission numbers were assigned to 151 predicted KEGG metabolic pathways. Digital abundance analysis using Illumina sequencing identified 78,124 transcripts among susceptible, resistant, herbicide-treated susceptible and herbicide-treated resistant barnyardgrass biotypes. From these analyses, eight herbicide target-site gene groups and four non-target-site gene groups were identified in the resistant biotype. These could be potential candidate genes involved in the herbicide resistance of barnyardgrass and could be used for further functional genomics research. C4 photosynthesis genes including RbcS, RbcL, NADP-me and MDH with complete CDS were identified using PCR and RACE technology.ConclusionsThis is the first large-scale transcriptome sequencing of E. crus-galli performed using the 454 GS-FLX platform. Potential candidate genes involved in the evolution of herbicide resistance were identified from the assembled sequences. This transcriptome data may serve as a reference for further gene expression and functional genomics studies, and will facilitate the study of herbicide resistance at the molecular level in this species as well as other weeds.

Dataset Information

Generation of a de novo transcriptome from equine lamellar tissue.

Publications

Generation of a de novo transcriptome from equine lamellar tissue.

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