This is a simple workflow to assist the inexperienced user with pre-processing, assembling and functionally annotating a de-novo transcriptome from RNA-seq data of non-model organisms. In order to successfully run all the steps, we recommend to use a linux computer with at least 8 cores. Please consider adapting file names and paths for your own use.

1. Software and data required

2. Quality control using FASTQC

3. Ribosomal RNA removal using SORTMERNA

4. Quality trimming and adapter removal using TRIMMOMATIC

5. Normalization using TRINITY

6. De-novo transcriptome assembly using TRINITY

7. Assembly validation using BOWTIE, BUSCO & BLASTX

8. Functional annotation using TRINOTATE

Montero-Mendieta, S., Grabherr, M., Lantz, H., De la Riva, I., Leonard, J.A., Webster, M.T., Vilà, C. (2017). A practical guide to build de-novo assemblies for single tissues of non-model organisms: the example of a Neotropical frog. PeerJ, 5, e3702. ​https://doi.org/10.7717/peerj.3702