title: Next generation sequencing and de novo transcriptomics to study gene evolution.
abstract: 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.
authors.affiliation: The University of Western Australia, School of Chemistry and Biochemistry & ARC Centre of Excellence in Plant Energy Biology, 35 Stirling Highway, Crawley Perth, 6009 Australia.
authors.forename: Achala S
authors.initials: AS
authors.lastname: Jayasena
authors.affiliation: The University of Western Australia, School of Chemistry and Biochemistry & ARC Centre of Excellence in Plant Energy Biology, 35 Stirling Highway, Crawley Perth, 6009 Australia.
authors.forename: David
authors.initials: D
authors.lastname: Secco
authors.affiliation: The University of Western Australia, School of Chemistry and Biochemistry & ARC Centre of Excellence in Plant Energy Biology, 35 Stirling Highway, Crawley Perth, 6009 Australia.
authors.forename: Kalia
authors.initials: K
authors.lastname: Bernath-Levin
authors.affiliation: The University of Western Australia, School of Chemistry and Biochemistry & ARC Centre of Excellence in Plant Energy Biology, 35 Stirling Highway, Crawley Perth, 6009 Australia ; The University of Western Australia, School of Plant Biology, 35 Stirling Highway, Crawley Perth, 6009 Australia.
authors.forename: Oliver
authors.initials: O
authors.lastname: Berkowitz
authors.affiliation: La Trobe University, Department of Botany, School of Life Sciences & ARC Centre of Excellence in Plant Energy Biology, AgriBio, the Centre for AgriBioscience, 5 Ring Road, Melbourne, Bundoora Victoria 3086 Australia.
authors.forename: James
authors.initials: J
authors.lastname: Whelan
authors.affiliation: The University of Western Australia, School of Chemistry and Biochemistry & ARC Centre of Excellence in Plant Energy Biology, 35 Stirling Highway, Crawley Perth, 6009 Australia.
authors.forename: Joshua S
authors.initials: JS
authors.lastname: Mylne
keywords: Cyclic peptides; De novo transcriptomics; Gene evolution; PawS1