Dataset Information

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.

SUBMITTER: Jayasena AS

PROVIDER: S-EPMC4216380 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Next generation sequencing and de novo transcriptomics to study gene evolution.

Jayasena Achala S AS Secco David D Bernath-Levin Kalia K Berkowitz Oliver O Whelan James J Mylne Joshua S JS

Plant methods 20141020 1

<h4>Background</h4>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.<h4>Results</h4>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 backg ...[more]

PMID: 25364374

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Project description:BACKGROUND:A strong focus of the post-genomic era is mining of the non-coding regulatory genome in order to unravel the function of regulatory elements that coordinate gene expression (Nat 489:57-74, 2012; Nat 507:462-70, 2014; Nat 507:455-61, 2014; Nat 518:317-30, 2015). Whole-genome approaches based on next-generation sequencing (NGS) have provided insight into the genomic location of regulatory elements throughout different cell types, organs and organisms. These technologies are now widespread and commonly used in laboratories from various fields of research. This highlights the need for fast and user-friendly software tools dedicated to extracting cis-regulatory information contained in these regulatory regions; for instance transcription factor binding site (TFBS) composition. Ideally, such tools should not require prior programming knowledge to ensure they are accessible for all users. RESULTS:We present TrawlerWeb, a web-based version of the Trawler_standalone tool (Nat Methods 4:563-5, 2007; Nat Protoc 5:323-34, 2010), to allow for the identification of enriched motifs in DNA sequences obtained from next-generation sequencing experiments in order to predict their TFBS composition. TrawlerWeb is designed for online queries with standard options common to web-based motif discovery tools. In addition, TrawlerWeb provides three unique new features: 1) TrawlerWeb allows the input of BED files directly generated from NGS experiments, 2) it automatically generates an input-matched biologically relevant background, and 3) it displays resulting conservation scores for each instance of the motif found in the input sequences, which assists the researcher in prioritising the motifs to validate experimentally. Finally, to date, this web-based version of Trawler_standalone remains the fastest online de novo motif discovery tool compared to other popular web-based software, while generating predictions with high accuracy. CONCLUSIONS:TrawlerWeb provides users with a fast, simple and easy-to-use web interface for de novo motif discovery. This will assist in rapidly analysing NGS datasets that are now being routinely generated. TrawlerWeb is freely available and accessible at: http://trawler.erc.monash.edu.au .

Dataset Information

Next generation sequencing and de novo transcriptomics to study gene evolution.

Publications

Next generation sequencing and de novo transcriptomics to study gene evolution.

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