Abstract
Alternative splicing generates multiple transcript and protein isoforms from the same gene and thus is important in gene expression regulation. To date, RNA-sequencing (RNA-seq) is the standard method for quantifying changes in alternative splicing on a genome-wide scale. Understanding the current limitations of RNA-seq is crucial for reliable analysis and the lack of high quality, comprehensive transcriptomes for most species, including model organisms such as Arabidopsis, is a major constraint in accurate quantification of transcript isoforms. To address this, we designed a novel pipeline with stringent filters and assembled a comprehensive Reference Transcript Dataset for Arabidopsis (AtRTD2) containing 82,190 non-redundant transcripts from 34 212 genes. Extensive experimental validation showed that AtRTD2 and its modified version, AtRTD2-QUASI, for use in Quantification of Alternatively Spliced Isoforms, outperform other available transcriptomes in RNA-seq analysis. This strategy can be implemented in other species to build a pipeline for transcript-level expression and alternative splicing analyses.
Biotechnology and Biological Sciences Research Council (BBSRC) [BB/K013661/1 and BB/K006568/1 to J.B.; BB/K006835/1 to H.N.]; Scottish Government Rural and Environment Science and Analytical Services division (RESAS) [to J.B. and R.Z.]; Austrian Science Fund (FWF) [P26333 to M.K., DK W1207 to A.B.]; LABEX Saclay Plant Sciences [to H.H.]; BBSRC EASTBIO PhD studentships (to N.T. and J.C.); European Alternative Splicing Network of Excellence (EURASNET) [LSHG-CT-2005-518238] for catalyzing important collaborations. Funding for open access charge: University of Dundee.