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Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks

A Corrigendum to this article was published on 25 September 2014

This article has been updated


Recent advances in high-throughput cDNA sequencing (RNA-seq) can reveal new genes and splice variants and quantify expression genome-wide in a single assay. The volume and complexity of data from RNA-seq experiments necessitate scalable, fast and mathematically principled analysis software. TopHat and Cufflinks are free, open-source software tools for gene discovery and comprehensive expression analysis of high-throughput mRNA sequencing (RNA-seq) data. Together, they allow biologists to identify new genes and new splice variants of known ones, as well as compare gene and transcript expression under two or more conditions. This protocol describes in detail how to use TopHat and Cufflinks to perform such analyses. It also covers several accessory tools and utilities that aid in managing data, including CummeRbund, a tool for visualizing RNA-seq analysis results. Although the procedure assumes basic informatics skills, these tools assume little to no background with RNA-seq analysis and are meant for novices and experts alike. The protocol begins with raw sequencing reads and produces a transcriptome assembly, lists of differentially expressed and regulated genes and transcripts, and publication-quality visualizations of analysis results. The protocol's execution time depends on the volume of transcriptome sequencing data and available computing resources but takes less than 1 d of computer time for typical experiments and 1 h of hands-on time.

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Figure 1: Software components used in this protocol.
Figure 2: An overview of the Tuxedo protocol.
Figure 3: Merging sample assemblies with a reference transcriptome annotation.
Figure 4: Analyzing groups of transcripts identifies differentially regulated genes.
Figure 5: CummeRbund helps users rapidly explore their expression data and create publication-ready plots of differentially expressed and regulated genes.
Figure 6: CummeRbund plots of the expression level distribution for all genes in simulated experimental conditions C1 and C2.
Figure 7: CummeRbund scatter plots highlight general similarities and specific outliers between conditions C1 and C2.
Figure 8
Figure 9: Differential analysis results for regucalcin.
Figure 10: Differential analysis results for Rala.

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Gene Expression Omnibus

Change history

  • 07 August 2014

     In the version of this article initially published, the computer script in Box 1 sections B and C, and in Procedure Step 1, contained errors: the last section of the final three lines of the script had ‘C1’ where it should have been ‘C2’, as follows: C1_R1_2.fq should have been C2_R1_2.fq C1_R2_2.fq should have been C2_R2_2.fq C1_R3_2.fq should have been C2_R3_2.fq Users are also directed to an official release version of Cufflinks (version 1.3.0) that produces nearly identical results to those shown in the manuscript, which were produced by Cufflinks 1.2.1 (an unofficial and undocumented development build that was the latest build available when the manuscript was originally written). The script in Procedure Step 16 and the data in Table 5 have been updated to reflect the output of version 1.3.0. The errors have been corrected in the HTML and PDF versions of the article.


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We are grateful to D. Hendrickson, M. Cabili and B. Langmead for helpful technical discussions. The TopHat and Cufflinks projects are supported by US National Institutes of Health grants R01-HG006102 (to S.L.S.) and R01-HG006129-01 (to L.P.). C.T. is a Damon Runyon Cancer Foundation Fellow. L.G. is a National Science Foundation Postdoctoral Fellow. A.R. is a National Science Foundation Graduate Research Fellow. J.L.R. is a Damon Runyon-Rachleff, Searle, and Smith Family Scholar, and is supported by Director's New Innovator Awards (1DP2OD00667-01). This work was funded in part by the Center of Excellence in Genome Science from the US National Human Genome Research Institute (J.L.R.). J.L.R. is an investigator of the Merkin Foundation for Stem Cell Research at the Broad Institute.

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Authors and Affiliations



C.T. is the lead developer for the TopHat and Cufflinks projects. L.G. designed and wrote CummeRbund. D.K., H.P. and G.P. are developers of TopHat. A.R. and G.P. are developers of Cufflinks and its accompanying utilities. C.T. developed the protocol, generated the example experiment and performed the analysis. L.P., S.L.S. and C.T. conceived the TopHat and Cufflinks software projects. C.T., D.R.K. and J.L.R. wrote the manuscript.

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Correspondence to Cole Trapnell.

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Trapnell, C., Roberts, A., Goff, L. et al. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc 7, 562–578 (2012).

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