title: 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.
authors.affiliation: Max-Delbrück-Center for Molecular Medicine, Berlin Institute for Medical Systems Biology, Robert Rössle Strasse 10, Berlin, Germany.
authors.forename: Catherine
authors.initials: C
authors.lastname: Adamidi
authors.forename: Yongbo
authors.initials: Y
authors.lastname: Wang
authors.forename: Dominic
authors.initials: D
authors.lastname: Gruen
authors.forename: Guido
authors.initials: G
authors.lastname: Mastrobuoni
authors.forename: Xintian
authors.initials: X
authors.lastname: You
authors.forename: Dominic
authors.initials: D
authors.lastname: Tolle
authors.forename: Matthias
authors.initials: M
authors.lastname: Dodt
authors.forename: Sebastian D
authors.initials: SD
authors.lastname: Mackowiak
authors.forename: Andreas
authors.initials: A
authors.lastname: Gogol-Doering
authors.forename: Pinar
authors.initials: P
authors.lastname: Oenal
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Rybak
authors.forename: Eric
authors.initials: E
authors.lastname: Ross
authors.forename: Alejandro
authors.initials: A
authors.lastname: Sánchez Alvarado
authors.forename: Stefan
authors.initials: S
authors.lastname: Kempa
authors.forename: Christoph
authors.initials: C
authors.lastname: Dieterich
authors.forename: Nikolaus
authors.initials: N
authors.lastname: Rajewsky
authors.forename: Wei
authors.initials: W
authors.lastname: Chen
mesh_terms: D000818:Animals; D002874:Chromosome Mapping; D003198:Computer Simulation; D020869:Gene Expression Profiling / Q000379:methods*; D016678:Genome; D017403:In Situ Hybridization; D058977:Molecular Sequence Annotation; D010932:Planarians / Q000235:genetics* / Q000378:metabolism; D020543:Proteome / Q000235:genetics / Q000378:metabolism; D040901:Proteomics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA / Q000379:methods*; D012984:Software; D013234:Stem Cells / Q000378:metabolism
chemical_list: D020543:Proteome; D012333:RNA, Messenger