Transcript Assembly and Quantification by RNA-Seq Reveals Unannotated Transcripts and Isoform Switching During Cell Differentiation

Overview

Journal Nat Biotechnol

Specialty Biotechnology

Date 2010 May 4

PMID 20436464

Citations 8503

Authors

Cole Trapnell

Brian A Williams

Geo Pertea

Ali Mortazavi

Gordon Kwan

Marijke J van Baren

Steven L Salzberg

Barbara J Wold

Lior Pachter

Affiliations

Soon will be listed here.

Abstract

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.

Citing Articles

A TaSnRK1α-TaCAT2 model mediates resistance to Fusarium crown rot by scavenging ROS in common wheat.

Yang X, Zhang L, Wei J, Liu L, Liu D, Yan X Nat Commun. 2025; 16(1):2549.

PMID: 40089587 DOI: 10.1038/s41467-025-57936-x.

Chromosome-level genome assembly of a critically endangered species Leuciscus chuanchicus.

Wang Q, Zhou Q, Liu H, Li J, Jiang Y Sci Data. 2025; 12(1):441.

PMID: 40089515 DOI: 10.1038/s41597-025-04787-2.

Interaction between the liver transcriptome and gut microbiota in mice during Toxoplasma gondii infection as identified by integrated transcriptomic and microbiome analysis.

Zou Y, Ma H, Yang X, Wei X, Chen C, Jiang J BMC Microbiol. 2025; 25(1):137.

PMID: 40087603 DOI: 10.1186/s12866-025-03852-5.

A systematic benchmark of Nanopore long-read RNA sequencing for transcript-level analysis in human cell lines.

Chen Y, Davidson N, Kei Wan Y, Yao F, Su Y, Gamaarachchi H Nat Methods. 2025; .

PMID: 40082608 DOI: 10.1038/s41592-025-02623-4.

GATA6 regulates WNT and BMP programs to pattern precardiac mesoderm during the earliest stages of human cardiogenesis.

Bisson J, Gordillo M, Kumar R, de Silva N, Yang E, Banks K Elife. 2025; 13.

PMID: 40080060 PMC: 11906159. DOI: 10.7554/eLife.100797.

References

Li B, Ruotti V, Stewart R, Thomson J, Dewey C . RNA-Seq gene expression estimation with read mapping uncertainty. Bioinformatics. 2009; 26(4):493-500. PMC: 2820677. DOI: 10.1093/bioinformatics/btp692. View

Yun K, Wold B . Skeletal muscle determination and differentiation: story of a core regulatory network and its context. Curr Opin Cell Biol. 1996; 8(6):877-89. DOI: 10.1016/s0955-0674(96)80091-3. View

Lareau L, Inada M, Green R, Wengrod J, Brenner S . Unproductive splicing of SR genes associated with highly conserved and ultraconserved DNA elements. Nature. 2007; 446(7138):926-9. DOI: 10.1038/nature05676. View

Eriksson N, Pachter L, Mitsuya Y, Rhee S, Wang C, Gharizadeh B . Viral population estimation using pyrosequencing. PLoS Comput Biol. 2008; 4(4):e1000074. PMC: 2323617. DOI: 10.1371/journal.pcbi.1000074. View

Cloonan N, Forrest A, Kolle G, Gardiner B, Faulkner G, Brown M . Stem cell transcriptome profiling via massive-scale mRNA sequencing. Nat Methods. 2008; 5(7):613-9. DOI: 10.1038/nmeth.1223. View

Mortazavi A, Williams B, McCue K, Schaeffer L, Wold B . Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods. 2008; 5(7):621-8. DOI: 10.1038/nmeth.1226. View

Pepke S, Wold B, Mortazavi A . Computation for ChIP-seq and RNA-seq studies. Nat Methods. 2009; 6(11 Suppl):S22-32. PMC: 4121056. DOI: 10.1038/nmeth.1371. View

Cordes K, Sheehy N, White M, Berry E, Morton S, Muth A . miR-145 and miR-143 regulate smooth muscle cell fate and plasticity. Nature. 2009; 460(7256):705-10. PMC: 2769203. DOI: 10.1038/nature08195. View

Yaffe D, Saxel O . A myogenic cell line with altered serum requirements for differentiation. Differentiation. 1977; 7(3):159-66. DOI: 10.1111/j.1432-0436.1977.tb01507.x. View

10.

Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N . The Sequence Alignment/Map format and SAMtools. Bioinformatics. 2009; 25(16):2078-9. PMC: 2723002. DOI: 10.1093/bioinformatics/btp352. View

11.

Griebel T, Zacher B, Ribeca P, Raineri E, Lacroix V, Guigo R . Modelling and simulating generic RNA-Seq experiments with the flux simulator. Nucleic Acids Res. 2012; 40(20):10073-83. PMC: 3488205. DOI: 10.1093/nar/gks666. View

12.

Hiller D, Jiang H, Xu W, Wong W . Identifiability of isoform deconvolution from junction arrays and RNA-Seq. Bioinformatics. 2009; 25(23):3056-9. PMC: 3167695. DOI: 10.1093/bioinformatics/btp544. View

13.

Cottle D, McGrath M, Cowling B, Coghill I, Brown S, Mitchell C . FHL3 binds MyoD and negatively regulates myotube formation. J Cell Sci. 2007; 120(Pt 8):1423-35. DOI: 10.1242/jcs.004739. View

14.

Langmead B, Trapnell C, Pop M, Salzberg S . Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009; 10(3):R25. PMC: 2690996. DOI: 10.1186/gb-2009-10-3-r25. View

15.

Bullard J, Purdom E, Hansen K, Dudoit S . Evaluation of statistical methods for normalization and differential expression in mRNA-Seq experiments. BMC Bioinformatics. 2010; 11:94. PMC: 2838869. DOI: 10.1186/1471-2105-11-94. View

16.

Jiang H, Wong W . Statistical inferences for isoform expression in RNA-Seq. Bioinformatics. 2009; 25(8):1026-32. PMC: 2666817. DOI: 10.1093/bioinformatics/btp113. View

17.

Wang E, Sandberg R, Luo S, Khrebtukova I, Zhang L, Mayr C . Alternative isoform regulation in human tissue transcriptomes. Nature. 2008; 456(7221):470-6. PMC: 2593745. DOI: 10.1038/nature07509. View

18.

Haas B, Delcher A, Mount S, Wortman J, Smith Jr R, Hannick L . Improving the Arabidopsis genome annotation using maximal transcript alignment assemblies. Nucleic Acids Res. 2003; 31(19):5654-66. PMC: 206470. DOI: 10.1093/nar/gkg770. View

19.

Endo T, Nadal-Ginard B . Transcriptional and posttranscriptional control of c-myc during myogenesis: its mRNA remains inducible in differentiated cells and does not suppress the differentiated phenotype. Mol Cell Biol. 1986; 6(5):1412-21. PMC: 367665. DOI: 10.1128/mcb.6.5.1412-1421.1986. View

20.

Denoeud F, Aury J, Da Silva C, Noel B, Rogier O, Delledonne M . Annotating genomes with massive-scale RNA sequencing. Genome Biol. 2008; 9(12):R175. PMC: 2646279. DOI: 10.1186/gb-2008-9-12-r175. View