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Abstract Background Accurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single-molecule long-read sequencing technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation, or incomplete cDNA synthesis. Results We present a new and comprehensive Arabidopsis thaliana Reference Transcript Dataset 3 (AtRTD3). AtRTD3 contains over 169,000 transcripts—twice that of the best current Arabidopsis transcriptome and including over 1500 novel genes. Seventy-eight percent of transcripts are from Iso-seq with accurately defined splice junctions and transcription start and end sites. We develop novel methods to determine splice junctions and transcription start and end sites accurately. Mismatch profiles around splice junctions provide a powerful feature to distinguish correct splice junctions and remove false splice junctions. Stratified approaches identify high-confidence transcription start and end sites and remove fragmentary transcripts due to degradation. AtRTD3 is a major improvement over existing transcriptomes as demonstrated by analysis of an Arabidopsis cold response RNA-seq time-series. AtRTD3 provides higher resolution of transcript expression profiling and identifies cold-induced differential transcription start and polyadenylation site usage. Conclusions AtRTD3 is the most comprehensive Arabidopsis transcriptome currently. It improves the precision of differential gene and transcript expression, differential alternative splicing, and transcription start/end site usage analysis from RNA-seq data. The novel methods for identifying accurate splice junctions and transcription start/end sites are widely applicable and will improve single-molecule sequencing analysis from any species. 

Alternative polyadenylation (APA) is a widespread post‐transcriptional mechanism that regulates gene expression throughmRNAmetabolism, playing a pivotal role in modulating phenotypic traits in rice (Oryza sativaL.). However, little is known about theAPA‐mediated regulation underlying the distinct characteristics between two major rice subspecies,indicaandjaponica. Using a poly(A)‐tag sequencing approach, polyadenylation (poly(A)) site profiles were investigated and compared pairwise from germination to the mature stage betweenindicaandjaponica, and extensive differentiation inAPAprofiles was detected genome‐wide. Genes with subspecies‐specific poly(A) sites were found to contribute to subspecies characteristics, particularly in disease resistance ofindicaand cold‐stress tolerance ofjaponica. In most tissues, differential usage ofAPAsites exhibited an apparent impact on the gene expression profiles between subspecies, and genes with those APA sites were significantly enriched in quantitative trait loci (QTL) related to yield traits, such as spikelet number and 1000‐seed weight. In leaves of the booting stage,APAsite‐switching genes displayed global shortening of 3′ untranslated regions with increased expression inindicacompared withjaponica, and they were overrepresented in the porphyrin and chlorophyll metabolism pathways. This phenomenon may lead to a higher chlorophyll content and photosynthesis inindicathan injaponica, being associated with their differential growth rates and yield potentials. We further constructed an online resource for querying and visualizing the poly(A) atlas in these two rice subspecies. Our results suggest thatAPAmay be largely involved in developmental differentiations between two rice subspecies, especially in leaf characteristics and the stress response, broadening our knowledge of the post‐transcriptional genetic basis underlying the divergence of rice traits.