Display Settings:

Format

Send to:

Choose Destination
See comment in PubMed Commons below
Nat Protoc. 2012 Mar 1;7(3):562-78. doi: 10.1038/nprot.2012.016.

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

Author information

  • 1Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. cole@broadinstitute.org

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.

PMID:
22383036
[PubMed - indexed for MEDLINE]
PMCID:
PMC3334321
Free PMC Article

Images from this publication.See all images (10)Free text

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Icon for Nature Publishing Group Icon for PubMed Central
    Loading ...
    Write to the Help Desk