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.

中文翻译：

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长读长RNA测序揭示了三刺刺鱼中广泛存在的性别特异性选择性剪接

替代亚型是真核生物表型多样性的重要贡献者。尽管短读长 RNA 测序增强了我们对异构体多样性的理解，但准确检测全长转录本具有挑战性，从而阻碍了许多替代异构体的识别。长读长测序技术使得对全长替代转录本进行测序、准确表征替代剪接事件、替代转录起始和终止位点以及 UTR 区域的差异成为可能。在这里，我们使用 Pacific Biosciences (PacBio) 长读长 RNA 测序 (Iso-Seq) 来检查三刺棘鱼 ( Gasterosteus aculeatus )（一种广泛使用的遗传模型物种）的五个器官的转录组。三刺刺鱼具有精细的基因组组装，其中基因注释基于短读长 RNA 测序和其他物种编码序列的预测。这表明一些现有注释可能不准确，或者替代转录本可能未得到充分表征。使用 Iso-Seq，我们检测到了数千种新的异构体，表明当前 Ensembl 基因注释中不存在许多异构体。此外，我们还完善了基因组内的许多现有注释。我们注意到许多位置不正确的转录起始位点通过长读长测序进行了细化。Iso-Seq 预测的转录起始位点更加准确，并通过 ATAC-seq 进行了验证。我们还检测到许多性别之间和跨器官的选择性剪接事件。我们在体细胞和性腺样本中发现了大量具有性别特异性亚型的基因。我们的研究强调了长读长测序在研究转录组复杂性方面的力量，极大地改善了三刺刺鱼的基因组资源。