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

Nat Protoc
Authors
Keywords
Abstract

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.

Year of Publication
2012
Journal
Nat Protoc
Volume
7
Issue
3
Pages
562-78
Date Published
2012 Mar 01
ISSN
1750-2799
URL
DOI
10.1038/nprot.2012.016
PubMed ID
22383036
PubMed Central ID
PMC3334321
Links
Grant list
R01 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