Long-read RNA sequencing reveals widespread sex-specific alternative splicing in threespine stickleback fish

Alternate isoforms are important contributors to phenotypic diversity across eukaryotes. Although short-read RNA-sequencing has increased our understanding of isoform diversity, it is challenging to accurately detect full-length tran-scripts, preventing the identification of many alternate isoforms. Long-read sequencing technologies have made it possible to sequence full-length alternative transcripts, accurately characterizing alternative splicing events, alternate transcription start and end sites, and differences in UTR regions. Here, we use Pacific Biosciences (PacBio) long-read RNA-sequencing (Iso-Seq) to examine the transcriptomes of five organs in threespine stickleback fish (Gasterosteus aculeatus), a widely used genetic model species. The threespine stickleback fish has a refined genome assembly in which gene annotations are based on short-read RNA sequencing and predictions from coding sequence of other species. This suggests some of the existing annotations may be inaccurate or alternative transcripts may not be fully characterized. Using Iso-Seq we detected thousands of novel isoforms, indicating many isoforms are absent in the current Ensembl gene annotations. In addition, we refined many of the existing annotations within the genome. We noted many improperly positioned transcription start sites that were refined with long-read sequencing. The Iso-Seq-predicted transcription start sites were more accurate and verified through ATAC-seq. We also detected many alternative splicing events between sexes and across organs. We found a substantial number of genes in both somatic and gonadal samples that had sex-specific isoforms. Our study highlights the power of long-read sequencing to study the complexity of transcriptomes, greatly improving genomic resources for the threespine stickleback fish.