可变剪接(AS)有助于物种之间的生物异质性,性别,组织,和细胞类型。许多疾病是由AS的改变或AS的改变引起的。因此,准确有效地测量AS对于评估分子表型至关重要,包括那些与疾病相关的。与短读取测序方法相比,长读取测序能够更准确地定量差异剪接的同工型表达。和第三代平台促进高通量实验。为了评估整个小脑的AS差异,皮质,海马体,和纹状体性别,我们生成并分析了牛津纳米孔技术(ONT)长读RNA测序(lrRNA-Seq)C57BL/6J小鼠脑cDNA文库。从超过8500万次通过质量控制指标的读数中,我们计算了差异基因表达(DGE),差异转录表达(DTE),以及不同大脑区域和性别的差异转录物使用(DTU)。我们发现了重要的DGE,DTE,和DTU跨大脑区域,小脑与其他三个区域相比差异最大。此外,我们发现了性别之间的区域特异性差异剪接,皮质中DTU的性别差异最大,海马中没有DTU。我们还报告了我们观察到的两种不同的性别DTU模式,性别分歧和性别特异性,在未来的研究中,这可能有助于解释各种神经和精神疾病患病率和预后的性别差异。最后,我们为研究人员构建了一个ShinyWeb应用程序,以进一步探索数据。我们的研究为社区提供了资源;它强调了AS在生物异质性中的重要性以及长读数测序对更好地了解大脑中的AS的实用性。
Alternative splicing (AS) contributes to the biological heterogeneity between species, sexes, tissues, and cell types. Many diseases are either caused by alterations in AS or by alterations to AS. Therefore, measuring AS accurately and efficiently is critical for assessing molecular phenotypes, including those associated with disease. Long-read sequencing enables more accurate quantification of differentially spliced isoform expression than short-read sequencing approaches, and third-generation platforms facilitate high-throughput experiments. To assess differences in AS across the cerebellum, cortex, hippocampus, and striatum by sex, we generated and analyzed Oxford Nanopore Technologies (ONT) long-read RNA sequencing (lrRNA-Seq) C57BL/6J mouse brain cDNA libraries. From > 85 million reads that passed quality control metrics, we calculated differential gene expression (DGE), differential transcript expression (DTE), and differential transcript usage (DTU) across brain regions and by sex. We found significant DGE, DTE, and DTU across brain regions and that the cerebellum had the most differences compared to the other three regions. Additionally, we found region-specific differential splicing between sexes, with the most sex differences in DTU in the cortex and no DTU in the hippocampus. We also report on two distinct patterns of sex DTU we observed, sex-divergent and sex-specific, that could potentially help explain sex differences in the prevalence and prognosis of various neurological and psychiatric disorders in future studies. Finally, we built a Shiny web application for researchers to explore the data further. Our study provides a resource for the community; it underscores the importance of AS in biological heterogeneity and the utility of long-read sequencing to better understand AS in the brain.