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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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.
The authors declare no competing financial interests.
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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). https://doi.org/10.1038/nprot.2012.016
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