Next generation sequencing and de novo transcriptomics to study gene evolution (Plant Methods)

Background: Studying gene evolution in non-model species by PCR-based approaches is limited to highly conserved genes. The plummeting cost of next generation sequencing enables the application of de novo transcriptomics to any species. Results: Here we describe how to apply de novo transcriptomics to pursue the evolution of a single gene of interest. We follow a rapidly evolving seed protein that encodes small, stable peptides. We use software that needs limited bioinformatics background and assemble four de novo seed transcriptomes. To demonstrate the quality of the assemblies, we confirm the predicted genes at the peptide level on one species which has over ten copies of our gene of interest. We explain strategies that favour assembly of low abundance genes, what assembly parameters help capture the maximum number of transcripts, how to develop a suite of control genes to test assembly quality and we compare several sequence depths to optimise cost and data volume. Conclusions: De novo transcriptomics is an effective approach for studying gene evolution in species for which genome support is lacking.