Abstract
Massively parallel sequencing of cDNA has enabled deep and efficient probing of transcriptomes. Current approaches for transcript reconstruction from such data often rely on aligning reads to a reference genome, and are thus unsuitable for samples with a partial or missing reference genome. Here we present the Trinity method for de novo assembly of full-length transcripts and evaluate it on samples from fission yeast, mouse and whitefly, whose reference genome is not yet available. By efficiently constructing and analyzing sets of de Bruijn graphs, Trinity fully reconstructs a large fraction of transcripts, including alternatively spliced isoforms and transcripts from recently duplicated genes. Compared with other de novo transcriptome assemblers, Trinity recovers more full-length transcripts across a broad range of expression levels, with a sensitivity similar to methods that rely on genome alignments. Our approach provides a unified solution for transcriptome reconstruction in any sample, especially in the absence of a reference genome.
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Acknowledgements
We thank L. Gaffney for help with figure preparation, J. Bochicchio for project management, the Broad Sequencing Platform for all sequencing work, A. Papanicolaou and M. Ott for Inchworm software testing and code enhancements, and F. Ribeiro for helpful discussions regarding error pruning. The work was supported in part by a grant from the National Human Genome Research Institute (NIH 1 U54 HG03067, Lander), the Howard Hughes Medical Institute, a National Institutes of Health PIONEER award, a Burroughs Wellcome Fund–Career Award at the Scientific Interface (A.R.), the US-Israel Binational Science Foundation (N.F. and A.R.), and funds from the National Institute of Allergy and Infectious Diseases under contract no. HHSN27220090018C. M.Y. was supported by a Clore Fellowship. K.L.-T. is a recipient of the European Young Investigator Award (EYRYI) funded by the European Science Foundation. A.R. is a researcher of the Merkin Foundation for Stem Cell Research at the Broad Institute.
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M.G.G., M.Y., B.J.H., K.L.-T., N.F. and A.R. conceived and designed the study. B.J.H., M.G.G. and M.Y. developed the Inchworm, Chrysalis and Butterfly components, respectively. N.R., F.D.P., B.W.B., C.N., K.L.-T. contributed to the study's conception and execution. J.Z.L., D.A.T., X.A., L.F., R.R., I.A., N.H., A.R. and A.G. designed and performed all experiments. Q.Z., Z.C. and E.M. contributed computational analyses. M.G.G., B.J.H. and M.Y. designed, implemented and evaluated all methods. A.R., N.F., M.G.G., B.J.H. and M.Y. wrote the manuscript, with input from all authors. A.R. and N.F. contributed equally to this paper.
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Supplementary Tables 1–3, Supplementary Methods, Supplementary Note and Supplementary Figures 1–9 (PDF 394 kb)
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Grabherr, M., Haas, B., Yassour, M. et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 29, 644–652 (2011). https://doi.org/10.1038/nbt.1883
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DOI: https://doi.org/10.1038/nbt.1883
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