De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis
Open accessAbstract
De novo assembly of RNA-seq data enables researchers to study transcriptomes without the need for a genome sequence; this approach can be usefully applied, for instance, in research on 'non-model organisms' of ecological and evolutionary importance, cancer samples or the microbiome. In this protocol we describe the use of the Trinity platform for de novo transcriptome assembly from RNA-seq data in non-model organisms. We also present Trinity-supported companion utilities for downstream applications, including RSEM for transcript abundance estimation, R/Bioconductor packages for identifying differentially expressed transcripts across samples and approaches to identify protein-coding genes. In the procedure, we provide a workflow for genome-independent transcriptome analysis leveraging the Trinity platform. The software, documentation and demonstrations are freely available from http://trinityrnaseq.sourceforge.net. The run time of this protocol is highly dependent on the size and complexity of data to be analyzed. The example data set analyzed in the procedure detailed herein can be processed in less than 5 h.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
Paired-end strand-specific sequencing and long reads are preferred for de novo transcriptome assembly because they are more informative.
“In general, PE strand-specific sequencing and long reads are preferred because they are more informative [33].”
From A survey of best practices for RNA-seq data analysis (2016) · cites this paper as [33]
Cites
Fast gapped-read alignment with Bowtie 2
Langmead, B., & Salzberg, S. L. (2012) · Nature Methods · doi:10.1038/nmeth.1923 · Cited by 56,400 · see citing works
Cutadapt removes adapter sequences from high-throughput sequencing reads
Martin, M. (2011) · EMBnet.journal · doi:10.14806/ej.17.1.200 · Cited by 33,889 · see citing works
Ultrafast and memory-efficient alignment of short DNA sequences to the human genome.
Langmead, B., Trapnell, C., Pop, M., & Salzberg, S. L. (2009) · Genome Biology · doi:10.1186/gb-2009-10-3-r25 · Cited by 21,849 · see citing works
Trapnell, C., Williams, B. A., Pertea, G., Mortazavi, A., Kwan, G., van Baren, M. J., Salzberg, S. L., Wold, B. J., & Pachter, L. (2010) · Nature Biotechnology · doi:10.1038/nbt.1621 · Cited by 15,556 · see citing works
Mapping and quantifying mammalian transcriptomes by RNA-Seq
Mortazavi, A., Williams, B. A., McCue, K., Schaeffer, L., & Wold, B. (2008) · Nature Methods · doi:10.1038/nmeth.1226 · Cited by 13,696 · see citing works
RNA-Seq: a revolutionary tool for transcriptomics
Wang, Z., Gerstein, M., & Snyder, M. (2009) · Nature Reviews Genetics · doi:10.1038/nrg2484 · Cited by 13,174 · see citing works
Bullard, J. H., Purdom, E., Hansen, K. D., & Dudoit, S. (2010) · BMC Bioinformatics · doi:10.1186/1471-2105-11-94 · Cited by 1,680 · see citing works
Differential expression in RNA-seq: A matter of depth
Tarazona, S., García-Alcalde, F., Dopazo, J., Ferrer, A., & Conesa, A. (2011) · Genome Research · doi:10.1101/gr.124321.111 · Cited by 1,632 · see citing works
Streaming fragment assignment for real-time analysis of sequencing experiments
Roberts, A., & Pachter, L. (2013) · Nature Methods · doi:10.1038/nmeth.2251 · Cited by 1,063 · see citing works
Comparative functional genomics of the fission yeasts
Rhind, N., Chen, Z., Yassour, M., Thompson, D. A., Haas, B. J., Habib, N., Wapinski, I., Roy, S., Lin, M. F., Heiman, D. I., et al. (2011) · Science · doi:10.1126/science.1203357 · Cited by 484 · see citing works
Self-citationStatistical Design and Analysis of RNA Sequencing Data
Auer, P. L., & Doerge, R. W. (2010) · Genetics · doi:10.1534/genetics.110.114983 · Cited by 393 · see citing works
Comparing de novo assemblers for 454 transcriptome data
Kumar, S., & Blaxter, M. L. (2010) · BMC Genomics · doi:10.1186/1471-2164-11-571 · Cited by 255 · see citing works
Flagged first, then by citations
Cited by
Sorted by citations
A survey of best practices for RNA-seq data analysis
Conesa, A., Madrigal, P., Tarazona, S., Gomez-Cabrero, D., Cervera, A., McPherson, A., Szcześniak, M. W., Gaffney, D. J., Elo, L. L., Zhang, X., et al. (2016) · Genome Biology · doi:10.1186/s13059-016-0881-8 · Cited by 2,794 · see citing works
GABA-modulating bacteria of the human gut microbiota
Strandwitz, P., Kim, K. H., Terekhova, D., Liu, J. K., Sharma, A., Levering, J., McDonald, D., Dietrich, D., Ramadhar, T. R., Lekbua, A., et al. (2018) · Nature Microbiology · doi:10.1038/s41564-018-0307-3 · Cited by 1,170 · see citing works
Llorens-Bobadilla, E., Zhao, S., Baser, A., Saiz-Castro, G., Zwadlo, K., & Martin-Villalba, A. (2015) · Cell Stem Cell · doi:10.1016/j.stem.2015.07.002 · Cited by 778 · see citing works
Haas, B. J., Dobin, A., Li, B., Stransky, N., Pochet, N., & Regev, A. (2019) · Genome biology · doi:10.1186/s13059-019-1842-9 · Cited by 646 · see citing works
Self-citationSimion, P., Philippe, H., Baurain, D., Jager, M., Richter, D. J., Di Franco, A., Roure, B., Satoh, N., Quéinnec, É., Ereskovsky, A., et al. (2017) · Current Biology · doi:10.1016/j.cub.2017.02.031 · Cited by 518 · see citing works
Muñoz, J. F., Gade, L., Chow, N. A., Loparev, V. N., Juieng, P., Berkow, E. L., Farrer, R. A., Litvintseva, A. P., & Cuomo, C. A. (2018) · Nature Communications · doi:10.1038/s41467-018-07779-6 · Cited by 458 · see citing works
Quiescence Modulates Stem Cell Maintenance and Regenerative Capacity in the Aging Brain
Kalamakis, G., Brüne, D., Ravichandran, S., Bolz, J., Fan, W., Ziebell, F., Stiehl, T., Catalá-Martinez, F., Kupke, J., Zhao, S., et al. (2019) · Cell · doi:10.1016/j.cell.2019.01.040 · Cited by 399 · see citing works
Error, signal, and the placement of Ctenophora sister to all other animals
Whelan, N. V., Kocot, K. M., Moroz, L. L., & Halanych, K. M. (2015) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1503453112 · Cited by 331 · see citing works
“Normalized Illumina reads were assembled using default parameters in Trinity v20131110 (46).”
MethodCtenophore relationships and their placement as the sister group to all other animals
Whelan, N. V., Kocot, K. M., Moroz, T. P., Mukherjee, K., Williams, P., Paulay, G., Moroz, L. L., & Halanych, K. M. (2017) · Nature Ecology & Evolution · doi:10.1038/s41559-017-0331-3 · Cited by 263 · see citing works
Genomic insights into the evolutionary origin of Myxozoa within Cnidaria
Chang, E. S., Neuhof, M., Rubinstein, N. D., Diamant, A., Philippe, H., Huchon, D., & Cartwright, P. (2015) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1511468112 · Cited by 262 · see citing works
Spiralian Phylogeny Informs the Evolution of Microscopic Lineages
Laumer, C. E., Bekkouche, N., Kerbl, A., Goetz, F., Neves, R. C., Sørensen, M. V., Kristensen, R. M., Hejnol, A., Dunn, C. W., Giribet, G., et al. (2015) · Current Biology · doi:10.1016/j.cub.2015.06.068 · Cited by 278 · see citing works
Kayal, E., Bentlage, B., Sabrina Pankey, M., Ohdera, A. H., Medina, M., Plachetzki, D. C., Collins, A. G., & Ryan, J. F. (2018) · BMC Evolutionary Biology · doi:10.1186/s12862-018-1142-0 · Cited by 255 · see citing works
Kean, R., Delaney, C., Sherry, L., Borman, A., Johnson, E. M., Richardson, M. D., Rautemaa-Richardson, R., Williams, C., & Ramage, G. (2018) · mSphere · doi:10.1128/msphere.00334-18 · Cited by 220 · see citing works
Borowiec, M. L., Lee, E. K., Chiu, J. C., & Plachetzki, D. C. (2015) · doi:10.1101/013946 · Cited by 172 · see citing works
The Blood Proteoform Atlas: A reference map of proteoforms in human hematopoietic cells
Melani, R. D., Gerbasi, V. R., Anderson, L. C., Sikora, J. W., Toby, T. K., Hutton, J. E., Butcher, D. S., Negrão, F., Seckler, H. S., Srzentić, K., et al. (2022) · Science · doi:10.1126/science.aaz5284 · Cited by 140 · see citing works
Self-citationYoulten, S. E., Kemp, J. P., Logan, J. G., Ghirardello, E. J., Sergio, C. M., Dack, M. R. G., Guilfoyle, S. E., Leitch, V. D., Butterfield, N. C., Komla-Ebri, D., et al. (2021) · Nature Communications · doi:10.1038/s41467-021-22517-1 · Cited by 133 · see citing works
Natural hybridization reveals incompatible alleles that cause melanoma in swordtail fish
Powell, D. L., García-Olazábal, M., Keegan, M., Reilly, P., Du, K., Díaz-Loyo, A. P., Banerjee, S., Blakkan, D., Reich, D., Andolfatto, P., et al. (2020) · Science · doi:10.1126/science.aba5216 · Cited by 124 · see citing works
Support for a clade of Placozoa and Cnidaria in genes with minimal compositional bias
Laumer, C. E., Gruber-Vodicka, H., Hadfield, M. G., Pearse, V. B., Riesgo, A., Marioni, J. C., & Giribet, G. (2018) · eLife · doi:10.7554/elife.36278 · Cited by 112 · see citing works
Cormier, A., Avia, K., Sterck, L., Derrien, T., Wucher, V., Andres, G., Monsoor, M., Godfroy, O., Lipinska, A., Perrineau, M., et al. (2017) · New Phytologist · doi:10.1111/nph.14321 · Cited by 103 · see citing works
Wang, X., Pipes, L., Trut, L. N., Herbeck, Y., Vladimirova, A. V., Gulevich, R. G., Kharlamova, A. V., Johnson, J. L., Acland, G. M., Kukekova, A. V., et al. (2018) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1800889115 · Cited by 100 · see citing works
Zhang, H., Du, H., & Xu, Y. (2021) · Applied and Environmental Microbiology · doi:10.1128/aem.02992-20 · Cited by 92 · see citing works
Muñoz, J. F., Delorey, T., Ford, C. B., Li, B. Y., Thompson, D. A., Rao, R. P., & Cuomo, C. A. (2019) · Nature Communications · doi:10.1038/s41467-019-09599-8 · Cited by 89 · see citing works
Self-citationBuckley, K. M., Ho, E. C. H., Hibino, T., Schrankel, C. S., Schuh, N. W., Wang, G., & Rast, J. P. (2017) · eLife · doi:10.7554/elife.23481 · Cited by 77 · see citing works
Diversity and evolution of myxozoan minicollagens and nematogalectins
Shpirer, E., Chang, E. S., Diamant, A., Rubinstein, N., Cartwright, P., & Huchon, D. (2014) · BMC Evolutionary Biology · doi:10.1186/s12862-014-0205-0 · Cited by 63 · see citing works
Li, J., Wang, J., Zhang, P., Wang, R., Mei, Y., Sun, Z., Fei, L., Jiang, M., Ma, L., E, W., et al. (2022) · Nature Genetics · doi:10.1038/s41588-022-01197-7 · Cited by 57 · see citing works
The Role of Homology and Orthology in the Phylogenomic Analysis of Metazoan Gene Content
Pett, W., Adamski, M., Adamska, M., Francis, W. R., Eitel, M., Pisani, D., & Wörheide, G. (2019) · Molecular Biology and Evolution · doi:10.1093/molbev/msz013 · Cited by 57 · see citing works
Transcriptome changes of blue-green algae, Arthrospira sp. in response to sulfate stress
Kumaresan, V., Nizam, F., Ravichandran, G., Viswanathan, K., Palanisamy, R., Bhatt, P., Arasu, M. V., Al-Dhabi, N. A., Mala, K., & Arockiaraj, J. (2017) · Algal Research · doi:10.1016/j.algal.2017.01.012 · Cited by 56 · see citing works
Unicellular origin of the animal microRNA machinery
Bråte, J., Neumann, R. S., Fromm, B., Haraldsen, A. A. B., Tarver, J. E., Suga, H., Donoghue, P. C. J., Peterson, K. J., Ruiz-Trillo, I., Grini, P. E., et al. (2018) · Current Biology · doi:10.1016/j.cub.2018.08.018 · Cited by 53 · see citing works
Red fox viromes in urban and rural landscapes
Campbell, S. J., Ashley, W., Gil-Fernandez, M., Newsome, T. M., Di Giallonardo, F., Ortiz-Baez, A. S., Mahar, J. E., Towerton, A. L., Gillings, M., Holmes, E. C., et al. (2020) · Virus Evolution · doi:10.1093/ve/veaa065 · Cited by 47 · see citing works
Nevado, B., Harris, S. A., Beaumont, M. A., & Hiscock, S. J. (2020) · Molecular Ecology · doi:10.1111/mec.15630 · Cited by 26 · see citing works
Evaluation of genetic demultiplexing of single-cell sequencing data from model species
Cardiello, J. F., Joven Araus, A., Giatrellis, S., Helsens, C., Simon, A., & Leigh, N. D. (2023) · Life Science Alliance · doi:10.26508/lsa.202301979 · Cited by 21 · see citing works
“11.0.simg /usr/local/bin/ trinityrnaseq/Analysis/SuperTranscripts/Trinity_gene_splice_ modeler.py –trinity_fasta Trinity_Pwal_v2.fasta –incl_malign – out_prefix out_dir A Notophthalmus de novo transcriptome from the study of Abdullayev et al (2013) was downloaded from https:// sandberglab.se/static/data/papers/redspot…”
InfluentialEcological engineering helps maximize function in algal oil production
Jackrel, S. L., Narwani, A., Bentlage, B., Levine, R. B., Hietala, D. C., Savage, P. E., Oakley, T. H., Denef, V. J., & Cardinale, B. J. (2018) · Applied and Environmental Microbiology · doi:10.1128/aem.00953-18 · Cited by 11 · see citing works
Darris, C. E., Tyus, J. E., Kelley, G., Ropelewski, A. J., Nicholas, H. B., Wang, X., & Nahashon, S. (2015) · BMC Genomics · doi:10.1186/s12864-015-1520-6 · Cited by 3 · see citing works
- Authors
- Haas, B. J., Papanicolaou, A., Yassour, M., Grabherr, M., Blood, P. D., Bowden, J., Couger, M. B., Eccles, D., Li, B., Lieber, M., MacManes, M. D., Ott, M., Orvis, J., Pochet, N., Strozzi, F., Weeks, N., Westerman, R., William, T., Dewey, C. N., Henschel, R., LeDuc, R. D., Friedman, N., & Regev, A.
- Affiliations
- Broad Institute (US)
- Commonwealth Scientific and Industrial Research Organisation (AU)
- Uppsala University (SE)
- Pittsburgh Supercomputing Center (US)
- Oklahoma State University (US)
- Griffith University (AU)
- University of Wisconsin–Madison (US)
- Center for Information Services and High Performance Computing (DE)
- QB3 (US)
- University of Maryland, Baltimore (US)
- Parco Tecnologico Padano (IT)
- Agricultural Research Service (US)
- Purdue University West Lafayette (US)
- GWT-TUD GmbH (Germany) (DE)
- Indiana University Bloomington (US)
- Hebrew University of Jerusalem (IL)
- Corresponding author
- Haas, Brian J
- Regev, Aviv
- ORCID
- Venue
- Nature Protocols
- Published
- 2013-07-11
- Volume
- 8
- Issue
- 8
- Pages
- 1494-1512
- ISSN
- 1750-2799 (ISSN-L)
- 1754-2189
- OpenAlex
- W2011657487
- Cited by
- 8,464
- References
- 12
- Citations by year
- 2026: 483 · 2025: 626 · 2024: 709 · 2023: 754 · 2022: 858 · 2021: 1021 · 2020: 887 · 2019: 812 · 2018: 708 · 2017: 634 · 2016: 469 · 2015: 338 · 2014: 138 · 2013: 12 · 2012: 1
- FWCI
- 142.07
- Type
- journal article
- Language
- en
- Open access
- green
- hdl.handle.net
- Licence
- Locations
- Nature Protocols (publishedVersion)
- PubMed (publishedVersion)
- DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc)
- Griffith Research Online (submittedVersion)
- Funders
- Howard Hughes Medical Institute
- National Human Genome Research Institute (R01HG005232, P50HG006193)
- National Institute of Allergy and Infectious Diseases (HHSN272200900018C)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Keywords
- de novo transcriptome assembly, Trinity, RNA-seq, non-model organisms, transcript abundance estimation, RSEM, differentially expressed transcripts
- MeSH terms
- Base Sequence, RNA, Schizosaccharomyces, Software, Sequence Analysis, RNA, Gene Expression Profiling, Schizosaccharomyces pombe Proteins, Transcriptome
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
