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Nat Protoc DOI:10.1038/nprot.2012.016

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

Publication TypeJournal Article
Year of Publication2012
AuthorsTrapnell, C, Roberts, A, Goff, L, Pertea, G, Kim, D, Kelley, DR, Pimentel, H, Salzberg, SL, Rinn, JL, Pachter, L
JournalNat Protoc
Date Published2012 Mar 01
KeywordsDNA, Complementary, Gene Expression Profiling, Genetic Association Studies, Genomics, Sequence Analysis, DNA, Software

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.


Alternate JournalNat Protoc
PubMed ID22383036
PubMed Central IDPMC3334321
Grant ListR01 HG006677 / HG / NHGRI NIH HHS / United States
R01-HG006102 / HG / NHGRI NIH HHS / United States
R01 HG006102-02 / HG / NHGRI NIH HHS / United States
R01 HG006677-12 / HG / NHGRI NIH HHS / United States
R01 GM083873 / GM / NIGMS NIH HHS / United States
R01 HG006102-01 / HG / NHGRI NIH HHS / United States
R01 HG006102 / HG / NHGRI NIH HHS / United States
R01 HG006677-13 / HG / NHGRI NIH HHS / United States
R01 HG006129 / HG / NHGRI NIH HHS / United States
P01 AR048929 / AR / NIAMS NIH HHS / United States
R01-HG006129-01 / HG / NHGRI NIH HHS / United States