A high resolution single molecule sequencing-based Arabidopsis transcriptome using novel methods of Iso-seq analysis

BackgroundAccurate and comprehensive annotations of transcript sequences are essential for accuracy of quantification of gene and transcript expression for analysis of differential gene and transcript expression, and transcriptional and post-transcriptional regulation. Single molecule long read technologies provide improved integrity of transcript structures including alternative splicing, transcription start and polyadenylation sites but accuracy is significantly affected by high sequencing errors.ResultsWe have generated a new and comprehensive Arabidopsis thaliana transcript reference dataset (AtRTD3) with extensive Iso-seq data from a range of plant material. We developed novel methods to determine splice junctions and transcription start and end sites accurately. Mis-match profiles around splice junctions provided a powerful and distinguishable feature between false and correct splice junctions allowing effective removal of falsely mapped splice junctions. Stratified approaches to remove transcript fragments due to degradation while taking account of expression abundance identified significant transcription start/end sites. The effectiveness of AtRTD3 was demonstrated by analysis of an RNA-seq time-series of plants exposed to cold which provided much higher resolution of transcript expression profiling and identified cold- and light-induced differential transcription start site and polyadenylation site usage.ConclusionsAtRTD3 is the most comprehensive, diverse and accurate Arabidopsis transcriptome available. It gives greater transcript quantification accuracy in RNA-seq data allowing detailed analysis of differential gene and transcript expression, alternative splicing and TSS and TES usage in Arabidopsis research. The novel methods for identifying splice junctions and transcription start/end sites are widely applicable and will improve single molecule sequencing analysis from any species.