De novo assembly and validation of planaria transcriptome by massive parallel sequencing and shotgun proteomics
ABSTRACT Freshwater planaria are a very attractive model system for stem cell biology, tissue homeostasis, and regeneration. The genome of the planarian Schmidtea mediterranea has recently been sequenced and is estimated to contain >20,000 protein-encoding genes. However, the characterization of its transcriptome is far from complete. Furthermore, not a single proteome of the entire phylum has been assayed on a genome-wide level. We devised an efficient sequencing strategy that allowed us to de novo assemble a major fraction of the S. mediterranea transcriptome. We then used independent assays and massive shotgun proteomics to validate the authenticity of transcripts. In total, our de novo assembly yielded 18,619 candidate transcripts with a mean length of 1118 nt after filtering. A total of 17,564 candidate transcripts could be mapped to 15,284 distinct loci on the current genome reference sequence. RACE confirmed complete or almost complete 5' and 3' ends for 22/24 transcripts. The frequencies of frame shifts, fusion, and fission events in the assembled transcripts were computationally estimated to be 4.2%-13%, 0%-3.7%, and 2.6%, respectively. Our shotgun proteomics produced 16,135 distinct peptides that validated 4200 transcripts (FDR ≤1%). The catalog of transcripts assembled in this study, together with the identified peptides, dramatically expands and refines planarian gene annotation, demonstrated by validation of several previously unknown transcripts with stem cell-dependent expression patterns. In addition, our robust transcriptome characterization pipeline could be applied to other organisms without genome assembly. All of our data, including homology annotation, are freely available at SmedGD, the S. mediterranea genome database.
Full-textDOI: · Available from: Pinar Önal, Aug 13, 2015
- SourceAvailable from: Alejandro Sánchez Alvarado
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- "We sought to define the molecular mechanisms driving pharynx regeneration by expression-profiling experiments. We designed custom oligonucleotide microarrays representing 43,806 predicted S. mediterranea transcripts and isoforms from various sources (Robb et al., 2008; Blythe et al., 2010; Adamidi et al., 2011). Based on our observations that pharynx regeneration triggered a localized stem cell proliferative response, we isolated a plug of tissue surrounding the pharynx wound site in order to enrich for those transcripts most directly relevant to this process (Figure 2F). "
ABSTRACT: Planarian flatworms regenerate every organ after amputation. Adult pluripotent stem cells drive this ability, but how injury activates and directs stem cells into the appropriate lineages is unclear. Here we describe a single-organ regeneration assay in which ejection of the planarian pharynx is selectively induced by brief exposure of animals to sodium azide. To identify genes required for pharynx regeneration, we performed an RNAi screen of 356 genes upregulated after amputation, using successful feeding as a proxy for regeneration. We found that knockdown of 20 genes caused a wide range of regeneration phenotypes and that RNAi of the forkhead transcription factor FoxA, which is expressed in a subpopulation of stem cells, specifically inhibited regrowth of the pharynx. Selective amputation of the pharynx therefore permits the identification of genes required for organ-specific regeneration and suggests an ancient function for FoxA-dependent transcriptional programs in driving regeneration. DOI: http://dx.doi.org/10.7554/eLife.02238.001eLife Sciences 04/2014; 3:e02238. DOI:10.7554/eLife.02238 · 8.52 Impact Factor
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- "rRNA-depleted RNA was selected by using the Ribo-ZeroTM rRNA removal kit following manufacturer's protocol (EpiCenter) and quantified using a Nanodrop 7500 spectrophotometer. 100 ng of rRNA-depleted RNA was fragmented and RNAseq library preparation was carried out as described previously (Adamidi et al., 2011). RNA-seq was performed on a HiSeq2000 sequencing platform with 1 Â 100 cycles of single read single-plex sequencing, in accordance with manufacturer's instructions (Illumina). "
ABSTRACT: Transcriptome analysis of polar bears (Ursus maritimus) yielded sequences with highest similarity to the human endogenous retrovirus group HERV-K(HML-2). Further analysis of the polar bear draft genome identified an endogenous betaretrovirus group comprising 26 proviral copies and 231 solo LTRs. Molecular dating indicates the group originated before the divergence of bears from a common ancestor but is not present in all carnivores. Closely related sequences were identified in the giant panda (Ailuropoda melanoleuca) and characterized from its genome. We have designated the polar bear and giant panda sequences U. maritimus endogenous retrovirus (UmaERV) and A. melanoleuca endogenous retrovirus (AmeERV), respectively. Phylogenetic analysis demonstrated that the bear virus group is nested within the HERV-K supergroup among bovine and bat endogenous retroviruses suggesting a complex evolutionary history within the HERV-K group. All individual remnants of proviral sequences contain numerous frameshifts and stop codons and thus, the virus is likely non-infectious.Virology 05/2013; 443(1). DOI:10.1016/j.virol.2013.05.008 · 3.28 Impact Factor
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- "(Sánchez Alvarado et al., 2002; Robb et al., 2008; Adamidi et al., 2011 "
ABSTRACT: Understanding the mechanisms governing the regeneration capabilities of many organisms is a fundamental interest in biology and medicine. An ever-increasing number of manipulation and molecular experiments are attempting to discover a comprehensive model for regeneration, with the planarian flatworm being one of the most important model species. Despite much effort, no comprehensive, constructive, mechanistic models exist yet, and it is now clear that computational tools are needed to mine this huge dataset. However, until now there is no database of regenerative experiments, and the current genotype-phenotype ontologies and databases are based on textual descriptions, which are not understandable by computers. To overcome these difficulties, we present here Planform (Planarian formalization), a manually-curated database and software tool for planarian regenerative experiments based on a mathematical graph formalism. The database contains more than a thousand experiments from the main publications in the planarian literature. The software tool provides the user with a graphical interface to easily interact with and mine the database. The presented system is a valuable resource for the regeneration community and, more importantly, will pave the way for the application of novel artificial intelligence tools to extract knowledge from this dataset. AVAILABILITY: The database and software tool are freely available at http://planform.daniel-lobo.com. CONTACT: email@example.com.Bioinformatics 02/2013; 29(8). DOI:10.1093/bioinformatics/btt088 · 4.62 Impact Factor