title: Genome-wide identification of transcript start and end sites by transcript isoform sequencing.
abstract: Hundreds of transcript isoforms with varying boundaries and alternative regulatory signals are transcribed from the genome, even in a genetically homogeneous population of cells. To study this transcriptional heterogeneity, we developed transcript isoform sequencing (TIF-seq), a method that allows the genome-wide profiling of full-length transcript isoforms defined by their exact 5' and 3' boundaries. TIF-seq entails the generation of full-length cDNA libraries, followed by their circularization and the sequencing of the junction fragments spanning the 5' and 3' transcript ends. By determining the respective co-occurrence of start and end sites of individual transcript molecules, TIF-seq can distinguish variations that conventional approaches for mapping single ends cannot, such as short abortive transcripts, bicistronic messages and overlapping transcripts that differ in lengths. The TIF-seq protocol we describe here can be applied to any eukaryotic organism (e.g., yeast, human), and it requires 6-10 d for generating TIF-seq libraries, 10 d for sequencing and 2-3 d for analysis.
authors: European Molecular Biology Laboratory (EMBL), Genome Biology Unit, Heidelberg, Germany.
authors: Vicent
authors: 000000029415788X
authors: V
authors: Pelechano
authors: 1] European Molecular Biology Laboratory (EMBL), Genome Biology Unit, Heidelberg, Germany. [2] Stanford Genome Technology Center, Palo Alto, California, USA.
authors: Wu
authors: W
authors: Wei
authors: European Molecular Biology Laboratory (EMBL), Genome Biology Unit, Heidelberg, Germany.
authors: Petra
authors: P
authors: Jakob
authors: 1] European Molecular Biology Laboratory (EMBL), Genome Biology Unit, Heidelberg, Germany. [2] Stanford Genome Technology Center, Palo Alto, California, USA. [3] Department of Genetics, Stanford University School of Medicine, Stanford, California, USA.
authors: Lars M
authors: LM
authors: Steinmetz
mesh_terms: D014644:Genetic Variation; D016678:Genome; D023281:Genomics; D020033:Protein Isoforms / Q000737:chemistry* / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017423:Sequence Analysis, RNA / Q000379:methods*
chemical_list: D020033:Protein Isoforms; D012333:RNA, Messenger