Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation.
High-throughput mRNA sequencing (RNA-Seq) promises simultaneous transcript discovery and abundance estimation. However, this would require algorithms that are not restricted by prior gene annotations and that account for alternative transcription and splicing. Here we introduce such algorithms in an open-source software program called Cufflinks. To test Cufflinks, we sequenced and analyzed >430 million paired 75-bp RNA-Seq reads from a mouse myoblast cell line over a differentiation time series. We detected 13,692 known transcripts and 3,724 previously unannotated ones, 62% of which are supported by independent expression data or by homologous genes in other species. Over the time series, 330 genes showed complete switches in the dominant transcription start site (TSS) or splice isoform, and we observed more subtle shifts in 1,304 other genes. These results suggest that Cufflinks can illuminate the substantial regulatory flexibility and complexity in even this well-studied model of muscle development and that it can improve transcriptome-based genome annotation.
Trapnell
Cole
C
Department of Computer Science, University of Maryland, College Park, Maryland, USA.
Williams
Brian A
BA
Pertea
Geo
G
Mortazavi
Ali
A
Kwan
Gordon
G
van Baren
Marijke J
MJ
Salzberg
Steven L
SL
Wold
Barbara J
BJ
Pachter
Lior
L
D000465:Algorithms; D000818:Animals; D002454:Cell Differentiation / Q000235:genetics*; D002460:Cell Line; D020869:Gene Expression Profiling / Q000379:methods*; D016678:Genome; D051379:Mice; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D020033:Protein Isoforms / Q000235:genetics* / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D017423:Sequence Analysis, RNA / Q000379:methods*; D012984:Software
D020033:Protein Isoforms; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger