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A robust (re-)annotation approach to generate unbiased mapping references for RNA-seq-based analyses of differential expression across closely related species.

ABSTRACT: BACKGROUND:RNA-seq based on short reads generated by next generation sequencing technologies has become the main approach to study differential gene expression. Until now, the main applications of this technique have been to study the variation of gene expression in a whole organism, tissue or cell type under different conditions or at different developmental stages. However, RNA-seq also has a great potential to be used in evolutionary studies to investigate gene expression divergence in closely related species. RESULTS:We show that the published genomes and annotations of the three closely related Drosophila species D. melanogaster, D. simulans and D. mauritiana have limitations for inter-specific gene expression studies. This is due to missing gene models in at least one of the genome annotations, unclear orthology assignments and significant gene length differences in the different species. A comprehensive evaluation of four statistical frameworks (DESeq2, DESeq2 with length correction, RPKM-limma and RPKM-voom-limma) shows that none of these methods sufficiently accounts for inter-specific gene length differences, which inevitably results in false positive candidate genes. We propose that published reference genomes should be re-annotated before using them as references for RNA-seq experiments to include as many genes as possible and to account for a potential length bias. We present a straight-forward reciprocal re-annotation pipeline that allows to reliably compare the expression for nearly all genes annotated in D. melanogaster. CONCLUSIONS:We conclude that our reciprocal re-annotation of previously published genomes facilitates the analysis of significantly more genes in an inter-specific differential gene expression study. We propose that the established pipeline can easily be applied to re-annotate other genomes of closely related animals and plants to improve comparative expression analyses.

SUBMITTER: Torres-Oliva M

PROVIDER: S-EPMC4877740 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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A robust (re-)annotation approach to generate unbiased mapping references for RNA-seq-based analyses of differential expression across closely related species.

Torres-Oliva Montserrat M Almudi Isabel I McGregor Alistair P AP Posnien Nico N

BMC genomics 20160524

<h4>Background</h4>RNA-seq based on short reads generated by next generation sequencing technologies has become the main approach to study differential gene expression. Until now, the main applications of this technique have been to study the variation of gene expression in a whole organism, tissue or cell type under different conditions or at different developmental stages. However, RNA-seq also has a great potential to be used in evolutionary studies to investigate gene expression divergence i ...[more]

PMID: 27220689

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Project description:Background: RNA-seq based on short reads generated by next generation sequencing technologies has become the main approach to study differential gene expression. Until now the main applications of this technique have been to study the variation of gene expression in a whole organism, tissue or cell type under different conditions or at different developmental stages. However, RNA-seq also has a great potential to be used in evolutionary studies to investigate gene expression divergence in closely related species. Since the more reliable statistical methods for differential gene expression inference are based on the use of raw read count data, the reference genomes of the species to be compared need to be highly comparable. Results: We show that the published genomes and annotations of the three closely related Drosophila species, D. melanogaster, D. simulans and D. mauritiana, have limitations for inter-specific gene expression studies. This is due to missing gene models in at least one of the genome annotations, unclear orthology assignments and significant length differences in the different species. We propose that published reference genomes should be re-annotated before using them as references for RNA-seq experiments to include as many genes as possible and to account for a potential length bias. For that we present a straight-forward reciprocal re-annotation pipeline that allows to reliably compare the expression for nearly all genes annotated in D. melanogaster. We carried out a RNA-seq experiment in combination with quantitative real-time PCR to confirm that the newly generated gene sets do not result in a high number of false positives as observed with references that still show a clear length difference of gene models between species. Conclusions: We conclude that our reciprocal re-annotation of previously published genomes facilitates the analysis of significantly more genes in an inter-specific differential gene expression study. We propose that the established pipeline can easily be applied to re-annotate other genomes of closely related animals and plants to improve comparative expression analyses.

Dataset Information

A robust (re-)annotation approach to generate unbiased mapping references for RNA-seq-based analyses of differential expression across closely related species.

Publications

A robust (re-)annotation approach to generate unbiased mapping references for RNA-seq-based analyses of differential expression across closely related species.

Similar Datasets

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