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.
At a glance
Gene Expression Omnibus
- Mapping and quantifying mammalian transcriptomes by RNA-seq. Nat. Methods 5, 621–628 (2008). , , , &
- Stem cell transcriptome profiling via massive-scale mRNA sequencing. Nat. Methods 5, 613–619 (2008). et al.
- The transcriptional landscape of the yeast genome defined by RNA sequencing. Science 320, 1344–1349 (2008). et al.
- The impact of next-generation sequencing technology on genetics. Trends Genet. 24, 133–141 (2008).
- Sequence identification of 2,375 human brain genes. Nature 355, 632–634 (1992). et al.
- Integrative annotation of human large intergenic noncoding RNAs reveals global properties and specific subclasses. Genes Dev. 25, 1915–1927 (2011). et al.
- Statistical inferences for isoform expression in RNA-seq. Bioinformatics 25, 1026–1032 (2009). &
- Transcript assembly and quantification by RNA-seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515 (2010). et al.
- A fast-acting reagent for accurate analysis of RNA secondary and tertiary structure by SHAPE chemistry. J. Am. Chem. Soc. 129, 4144–4145 (2007). &
- RNA-seq gene expression estimation with read mapping uncertainty. Bioinformatics 26, 493–500 (2010). , , , &
- RNA-seq: an assessment of technical reproducibility and comparison with gene expression arrays. Genome Res. 18, 1509–1517 (2008). , , , &
- Computational methods for transcriptome annotation and quantification using RNA-seq. Nat. Methods 8, 469–477 (2011). , , &
- TopHat: discovering splice junctions with RNA-seq. Bioinformatics 25, 1105–1111 (2009). , &
- Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells. Nature 470, 68–73 (2011). et al.
- The developmental transcriptome of Drosophila melanogaster. Nature 471, 473–479 (2011). et al.
- Whole transcriptome sequencing reveals gene expression and splicing differences in brain regions affected by Alzheimer's disease. PLoS ONE 6, e16266 (2011). , , &
- Massive parallel sequencing of mRNA in identification of unannotated salinity stress-inducible transcripts in rice (Oryza sativa L.). BMC Genomics 11, 683 (2010). et al.
- Galaxy Team Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol. 11, R86 (2010). , &
- Fast and SNP-tolerant detection of complex variants and splicing in short reads. Bioinformatics 26, 873–881 (2010). &
- MapSplice: accurate mapping of RNA-seq reads for splice junction discovery. Nucleic Acids Res. 38, e178 (2010). et al.
- Detection of splice junctions from paired-end RNA-seq data by SpliceMap. Nucleic Acids Res. 38, 4570–4578 (2010). , , , &
- Ab initio reconstruction of cell type-specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincRNAs. Nat. Biotechnol. 28, 503–510 (2010). et al.
- Alternative expression analysis by RNA sequencing. Nat. Methods 7, 843–847 (2010). et al.
- Analysis and design of RNA sequencing experiments for identifying isoform regulation. Nat. Methods 7, 1009–1015 (2010). , , &
- Estimation of alternative splicing isoform frequencies from RNA-seq data. Algorithms Mol. Biol. 6, 9 (2011). , , &
- Differential expression analysis for sequence count data. Genome Biol. 11, R106 (2010). &
- edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2009). , &
- DEGseq: an R package for identifying differentially expressed genes from RNA-seq data. Bioinformatics 26, 136–138 (2010). , , , &
- Full-length transcriptome assembly from RNA-seq data without a reference genome. Nat. Biotechnol. 29, 644–652 (2011). et al.
- De novo assembly and analysis of RNA-seq data. Nat. Methods 7, 909–912 (2010). et al.
- Genome-wide mapping of in vivo protein-DNA interactions. Science 316, 1497–1502 (2007). , , &
- Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling. Science 324, 218–223 (2009). , , &
- Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009). , , &
- An experimental study of a compressed index. Information Sci. 135, 13–28 (2001). &
- Identification of novel transcripts in annotated genomes using RNA-seq. Bioinformatics 27, 2325–2329 (2011). , , &
- Modeling non-uniformity in short-read rates in RNA-seq data. Genome Biol. 11, R50 (2010). , &
- Biases in Illumina transcriptome sequencing caused by random hexamer priming. Nucleic Acids Res. 38, e131 (2010). , &
- Improving RNA-seq expression estimates by correcting for fragment bias. Genome Biol. 12, R22 (2011). , , , &
- Comprehensive comparative analysis of strand-specific RNA sequencing methods. Nat. Methods 7, 709–715 (2010). et al.
- Sequencing technology does not eliminate biological variability. Nat. Biotechnol. 29, 572–573 (2011). , , &
- ggplot2: Elegant Graphics for Data Analysis (Use R) p 224 (Springer, 2009).
- Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011). et al.
- The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009). et al.
- Cloud computing and the DNA data race. Nat. Biotechnol. 28, 691–693 (2010). , &