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Ab initio reconstruction of cell type–specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincRNAs

Nature Biotechnology volume 28, pages 503–510 (2010)Cite this article

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A Corrigendum to this article was published on 01 July 2010

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Abstract

Massively parallel cDNA sequencing (RNA-Seq) provides an unbiased way to study a transcriptome, including both coding and noncoding genes. Until now, most RNA-Seq studies have depended crucially on existing annotations and thus focused on expression levels and variation in known transcripts. Here, we present Scripture, a method to reconstruct the transcriptome of a mammalian cell using only RNA-Seq reads and the genome sequence. We applied it to mouse embryonic stem cells, neuronal precursor cells and lung fibroblasts to accurately reconstruct the full-length gene structures for most known expressed genes. We identified substantial variation in protein coding genes, including thousands of novel 5′ start sites, 3′ ends and internal coding exons. We then determined the gene structures of more than a thousand large intergenic noncoding RNA (lincRNA) and antisense loci. Our results open the way to direct experimental manipulation of thousands of noncoding RNAs and demonstrate the power of ab initio reconstruction to render a comprehensive picture of mammalian transcriptomes.

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Figure 1: Scripture: a method for ab initio transcriptome reconstruction from RNA-Seq data.
Figure 2: Scripture correctly reconstructs full-length transcripts for most annotated protein coding genes.
Figure 3: Alternative 5′ ends, 3′ ends and novel coding exons in transcripts reconstructed by Scripture.
Figure 4: Noncoding transcripts reconstructed by Scripture.
Figure 5: Protein coding capacity, conservation levels and expression of lincRNAs and multi-exonic antisense transcripts.

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Change history

  • 09 July 2010

    In the version of this article initially published, the fourth sentence in the methods section “RNA extraction and library preparation” instead of saying a “procedure that combines a random priming step with a shearing step8,9,28 and results in fragments of ~700 bp in size” should have read, “procedure that combines fragmentation of mRNA to a peak size of ~750 nucleotides by heating6 followed by random-primed reverse transcription8.”. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank M. Wernig (MIT) for providing NPC; M. Lin and M. Kellis (MIT) for CSF code; the Broad Sequencing Platform for sample sequencing; L. Gaffney for assistance with graphics; and C. Burge, J. Merkin, R. Bradley and members of Lander and Regev laboratories—in particular, M. Yassour, T. Mikkelsen and I. Amit—for discussions. A.R. and J.L.R. were supported by the Merkin Family Foundation for Stem Cell Research at the Broad Institute. M. Guttman was supported by a Vertex scholarship. Work was supported by a Burroughs Wellcome Fund Career Award at the Scientific Interface, a US National Institutes of Health PIONEER award, a US National Human Genome Research Institute (NHGRI) R01 grant and the Howard Hughes Medical Institute (A.R.), and NHGRI and the Broad Institute of MIT and Harvard (E.S.L.).

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Author notes

  1. Mitchell Guttman and Manuel Garber: These authors contributed equally to this work.

Authors and Affiliations

  1. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA

    Mitchell Guttman, Manuel Garber, Joshua Z Levin, Julie Donaghey, James Robinson, Xian Adiconis, Lin Fan, Magdalena J Koziol, Andreas Gnirke, Chad Nusbaum, John L Rinn, Eric S Lander & Aviv Regev

  2. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Mitchell Guttman, Eric S Lander & Aviv Regev

  3. Department of Pathology, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USA

    Magdalena J Koziol & John L Rinn

  4. Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA

    Eric S Lander

  5. Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Aviv Regev

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Contributions

M. Guttman and M. Garber conceived the project, designed research, implemented Scripture, performed computational analysis and wrote the paper. A.G., C.N. and J.Z.L. oversaw cDNA sequencing, provided molecular biology advice and helped to edit the manuscript. J.D. constructed cDNA libraries, performed validation experiments and helped to edit the manuscript. J.R. implemented components of Scripture and provided computational support and technical advice. X.A., L.F. and M.J.K. constructed cDNA libraries. J.L.R. provided reagents and helped edit the manuscript. E.S.L. designed research direction and wrote the paper. A.R. provided cDNA sequencing guidance, conceived the project, designed research direction and wrote the paper.

Corresponding authors

Correspondence to Mitchell Guttman, Manuel Garber or Aviv Regev.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Notes 1 and 2, Supplementary Figures 1–7 (PDF 3117 kb)

Supplementary Table 1

Number of novel transcriptional events in ES, MLF and NPC (XLS 10 kb)

Supplementary Table 2

Primer sequences used for validation of novel events (XLS 13 kb)

Supplementary Software

scripture.jar scripture.src.tgz (ZIP 15384 kb)

Supplementary Data

ES.gff.gz ESTranscriptGraphs.tar.gz (ZIP 37695 kb)

Supplementary Data

MLF.gff.gz MLFTranscriptGraphs.tar.gz (ZIP 14834 kb)

Supplementary Data

NPC.gff.gz NPCTranscriptGraphs.tar.gz (ZIP 42407 kb)

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Guttman, M., Garber, M., Levin, J. et al. Ab initio reconstruction of cell type–specific transcriptomes in mouse reveals the conserved multi-exonic structure of lincRNAs. Nat Biotechnol 28, 503–510 (2010). https://doi.org/10.1038/nbt.1633

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