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.
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- Supplementary Note (699 KB)
Supplementary materials for de novo transcript sequence reconstruction from RNA-seq: reference generation and analysis with Trinity.
- Supplementary Figure 1 (554 KB)
Defining minimum edge thresholds during initial Butterfly graph pruning.
- Supplementary Figure 2 (551 KB)
Butterfly's minimum support requirement for path extension during transcript reconstruction.
- Supplementary Figure 3 (530 KB)
Merging of insufficiently different path sequences.
- Supplementary Figure 4 (536 KB)
Enforcing path restrictions via triplet locking.
- Supplementary Figure 5 (540 KB)
Restrictions on the number of paths to be extended at each node.
- Supplementary Figure 6 (636 KB)
Evaluating assembly completeness for the S. pombe transcriptome.
- Supplementary Figure 7 (584 KB)
Evaluating assembly completeness for the mouse dendritic cell transcriptome.
- Supplementary Figure 8 (551 KB)
Correlation of expression values between reference transcripts and Trinity transcript components according to percent length agreement in S. pombe.
- Supplementary Figure 9 (584 KB)
Agreement between expression profiles calculated based on reference transcripts and trinity components at different S. pombe samples.