Composite transcriptome assembly of RNA-seq data in a sheep model for delayed bone healing

Abstract

BackgroundThe sheep is an important model organism for many types of medically relevant research, but molecular genetic experiments in the sheep have been limited by the lack of knowledge about ovine gene sequences.ResultsPrior to our study, mRNA sequences for only 1,556 partial or complete ovine genes were publicly available. Therefore, we developed a composite de novo transcriptome assembly method for next-generation sequence data to combine known ovine mRNA and EST sequences, mRNA sequences from mouse and cow, and sequences assembled de novo from short read RNA-Seq data into a composite reference transcriptome, and identified transcripts from over 12 thousand previously undescribed ovine genes. Gene expression analysis based on these data revealed substantially different expression profiles in standard versus delayed bone healing in an ovine tibial osteotomy model. Hundreds of transcripts were differentially expressed between standard and delayed healing and between the time points of the standard and delayed healing groups. We used the sheep sequences to design quantitative RT-PCR assays with which we validated the differential expression of 26 genes that had been identified by RNA-seq analysis. A number of clusters of characteristic expression profiles could be identified, some of which showed striking differences between the standard and delayed healing groups. Gene Ontology (GO) analysis showed that the differentially expressed genes were enriched in terms including extracellular matrix, cartilage development, contractile fiber, and chemokine activity.ConclusionsOur results provide a first atlas of gene expression profiles and differentially expressed genes in standard and delayed bone healing in a large-animal model and provide a number of clues as to the shifts in gene expression that underlie delayed bone healing. In the course of our study, we identified transcripts of 13,987 ovine genes, including 12,431 genes for which no sequence information was previously available. This information will provide a basis for future molecular research involving the sheep as a model organism.

A Polymerase Chain Reaction-Based Method for Isolating Clones from a Complimentary DNA Library in Sheep

“…As it happened, the mRNA had also been isolated in sheep by Jager et al 25 but was wrongly annotated as CEP290, the 3¢UTR of which, in all vertebrates, abuts the 3¢UTR of C12orf29 on the opposite strand. The C12orf29 sequence from that study contained an atypical poly-adenylation signal (AATTAAA), similar to what we had identified in our clone; this feature was therefore unlikely to be an artifact.…”

“…25 Studies such as these generate large data sets that require high-performance computing infrastructure and specialized bioinformaticians to assemble and analyze. The study by Jager et al, 25 for example, analyzed over 830,000 contigs with an average length of 134 bp using the preassembly program Velvet, which were then extended using the de novo transcriptome assembly program Oases. This two-step approach yielded a total of 117,594 extended contigs with an average length of 1374 bp.…”

The Interaction of BMP2‐Induced Defect Healing in Rat and Fixator Stiffness Modulates Matrix Alignment and Contraction

“…In previous studies, we observed differential expression of genes involved in cellular contraction in large and small animal models of bone defect healing 31, 45. Based on the results of the exploratory analyses described here, we performed a targeted in vitro experiment.…”

“…The interplay between mechanical and biological stimuli in vivo to gate either woven or lamellar bone formation has been demonstrated in mechanical loaded intact bone27, 28, 29 and distraction osteogenesis 30. Our group investigated this interaction in osteotomized bone 31. Additionally, we treated a rat critical‐sized defect with a high dose of BMP2 only and in combination with weekly controlled axial compressive mechanical loading.…”

PERSPECTIVES Transcriptome sequencing: next generation approach to RNA functional analysis

“…Thus, the use of de novo assembly significantly raises sequencing costs. Despite these disadvantages, successful de novo transcriptome assembly has been recently performed for lower and higher eukaryotes (Martin et al, 2010;Garg et al, 2011;Grabherr et al, 2011;Jäger et al, 2011). Often, the two strategies (reference genome-based and de novo) are combined by first mapping the data to the reference sequence and further de novo assembly of reads that failed to align.…”