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 transcripts, 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.

Background: Pronounced asymmetric changes in ocular globe size during eye development have been observed in a number of species ranging from humans to lizards. In contrast, largely symmetric changes in globe size have been described for other species like rodents. We propose that asymmetric changes in the three-dimensional structure of the developing eye correlate with the types of retinal remodeling needed to produce areas of high photoreceptor density. To test this idea, we systematically examined three-dimensional aspects of globe size as a function of eye development in the bifoveated brown anole, Anolis sagrei.Results: During embryonic development, the anole eye undergoes dynamic changes in ocular shape. Initially spherical, the eye elongates in the presumptive foveal regions of the retina and then proceeds through a period of retraction that returns the eye to its spherical shape. During this period of retraction, pit formation and photoreceptor cell packing are observed. We found a similar pattern of elongation and retraction associated with the single fovea of the veiled chameleon, Chamaeleo calyptratus.Conclusions: These results, together with those reported for other foveated species, support the idea that areas of high photoreceptor packing occur in regions where the ocular globe asymmetrically elongates and retracts during development.

Background: Pronounced asymmetric changes in ocular globe size during eye development have been observed in a number of species ranging from humans to lizards. In contrast, largely symmetric changes in globe size have been described for other species such as rodents. We propose that asymmetric changes in the three-dimensional structure of the developing eye correlate with the types of retinal remodeling needed to produce areas of high photoreceptor density. As a test of this idea, we systematically examined three-dimensional aspects of globe size as a function of eye development in the bifoveated brown anole, Anolis sagrei. Results: During embryonic development, the anole eye undergoes dynamic changes in ocular shape. Initially spherical, the eye elongates in the presumptive foveal regions of the retina and then proceeds through a period of retraction that returns the eye to its spherical shape. During this period of retraction, pit formation and photoreceptor cell packing are observed. We found a similar pattern of elongation and retraction associated with the single fovea of the veiled chameleon, Chamaeleo calyptratus. Conclusions: These results, together with those reported for other foveated species, support the idea that areas of high photoreceptor packing occur in regions where the ocular globe asymmetrically elongates and retracts during development.

Alternate isoforms contribute immensely to phenotypic diversity across eukaryotes. While short read RNA-sequencing has increased our understanding of isoform diversity, it is challenging to accurately detect full-length transcripts, 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 utilize PacBio long read RNA-sequencing (Iso-Seq) to examine the transcriptomes of five tissues in threespine stickleback fish (Gasterosteus aculeatus), a widely used genetic model species. The threespine stickleback fish has a refined genome assembly with gene annotations that 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, verified through ATAC-seq. We were also able to detect many alternative splicing events between sexes and across tissues. We found a substantial number of genes in both somatic and gonad tissue 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.