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Differential analysis of gene regulation at transcript resolution with RNA-seq

Nature Biotechnology volume 31, pages 46–53 (2013)Cite this article

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Abstract

Differential analysis of gene and transcript expression using high-throughput RNA sequencing (RNA-seq) is complicated by several sources of measurement variability and poses numerous statistical challenges. We present Cuffdiff 2, an algorithm that estimates expression at transcript-level resolution and controls for variability evident across replicate libraries. Cuffdiff 2 robustly identifies differentially expressed transcripts and genes and reveals differential splicing and promoter-preference changes. We demonstrate the accuracy of our approach through differential analysis of lung fibroblasts in response to loss of the developmental transcription factor HOXA1, which we show is required for lung fibroblast and HeLa cell cycle progression. Loss of HOXA1 results in significant expression level changes in thousands of individual transcripts, along with isoform switching events in key regulators of the cell cycle. Cuffdiff 2 performs robust differential analysis in RNA-seq experiments at transcript resolution, revealing a layer of regulation not readily observable with other high-throughput technologies.

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Figure 1: Changes in fragment count for a gene does not necessarily equal a change in expression.
Figure 2: An overview of the Cuffdiff 2 approach to isoform-level differential analysis of RNA-seq data.
Figure 3: Comparison of Cuffdiff 2 with other expression platforms.
Figure 4: Accuracy of Cuffdiff 2 over varied experimental designs.
Figure 5: Changes in expression of cell cycle regulatory genes in response to HOXA1 knockdown.
Figure 6: Cell cycle analysis after HOXA1 knockdown.

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Acknowledgements

We are grateful to D. Kelley for a careful reading of the manuscript, and B. Wold for sharing the hESC RNA-seq data. We are also thankful for the ongoing development efforts of A. Roberts, B. Langmead, D. Kim, G. Pertea, H. Pimentel and S. Salzberg. C.T. and D.G.H. are Damon Runyon Postdoctoral Fellows. J.L.R. is a Damon Runyon-Rachleff Inovator fellow. This work was supported by US National Institutes of Health grants DP2OD006670, P01GM099117, P50HG006193 and RO1ES020260 (to J.L.R.) and R01 HG006129 and R01 DK094699 (to L.P.).

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Author notes

  1. Cole Trapnell and David G Hendrickson: These authors contributed equally to this work.

  2. John L Rinn and Lior Pachter: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA

    Cole Trapnell, David G Hendrickson, Martin Sauvageau, Loyal Goff & John L Rinn

  2. The Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, Massachusetts, USA

    Cole Trapnell, David G Hendrickson, Martin Sauvageau, Loyal Goff & John L Rinn

  3. Computer Science and Artificial Intelligence Laboratory, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Loyal Goff

  4. Department of Mathematics, University of California Berkeley, Berkeley, California, USA

    Lior Pachter

  5. Department of Molecular & Cell Biology, University of California Berkeley, California, USA

    Lior Pachter

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  1. Cole Trapnell
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Contributions

C.T. and L.P. developed the mathematics and statistics. D.G.H. and M.S. performed the experiments. D.G.H. and C.T. designed the experiments and performed the analysis. C.T. and L.G. implemented the software. L.P., J.L.R., D.G.H. and C.T. conceived the research. All authors wrote and approved the manuscript.

Corresponding authors

Correspondence to John L Rinn or Lior Pachter.

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The authors declare no competing financial interests.

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Trapnell, C., Hendrickson, D., Sauvageau, M. et al. Differential analysis of gene regulation at transcript resolution with RNA-seq. Nat Biotechnol 31, 46–53 (2013). https://doi.org/10.1038/nbt.2450

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