Helmholtz Gemeinschaft

Search
Browse
Statistics
Feeds

De novo assembly and validation of planaria transcriptome by massive parallel sequencing and shotgun proteomics

[img] PDF - Requires a PDF viewer such as GSview, Xpdf or Adobe Acrobat Reader
441kB

Official URL:https://doi.org/10.1101/gr.113779.110
PubMed:View item in PubMed
Creators Name:Adamidi, C. and Wang, Y. and Gruen, D. and Mastrobuoni, G. and You, X. and Tolle, D. and Dodt, M. and Mackowiak, S.D. and Gogol-Doering, A. and Oenal, P. and Rybak, A. and Ross, E. and Alvarado, A.S. and Kempa, S. and Dieterich, C. and Rajewsky, N. and Chen, W.
Journal Title:Genome Research
Journal Abbreviation:Genome Res
Volume:21
Number:7
Page Range:1193-1200
Date:July 2011
Keywords:Chromosome Mapping, Computer Simulation, DNA Sequence Analysis, Gene Expression Profiling, Genome, In Situ Hybridization, Messenger RNA, Molecular Sequence Annotation, Planarians, Proteome, Proteomics, Software, Stem Cells, Animals
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.
ISSN:1088-9051
Publisher:Cold Spring Harbor Laboratory Press (U.S.A.)
Item Type:Article

Repository Staff Only: item control page

Downloads

Downloads per month over past year

Open Access
MDC Library