RNA isoform diversity, produced via alternative splicing, and alternative usage of transcription start and poly(A) sites, results in varied transcripts being derived from the same gene. Distinct isoforms can play important biological roles, including by changing the sequences or expression levels of protein products. The first single-cell approaches to RNA sequencing—and later, spatial approaches—which are now widely used for the identification of differentially expressed genes, rely on short reads and offer the ability to transcriptomically compare different cell types but are limited in their ability to measure differential isoform expression. More recently, long-read sequencing methods have been combined with single-cell and spatial technologies in order to characterize isoform expression. In this review, we provide an overview of the emergence of single-cell and spatial long-read sequencing and discuss the challenges associated with the implementation of these technologies and interpretation of these data. We discuss the opportunities they offer for understanding the relationships between the distinct variable elements of transcript molecules and highlight some of the ways in which they have been used to characterize isoforms’ roles in development and pathology. Single-nucleus long-read sequencing, a special case of the single-cell approach, is also discussed. We attempt to cover both the limitations of these technologies and their significant potential for expanding our still-limited understanding of the biological roles of RNA isoforms.

中文翻译：

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通过将长读长测序与单细胞和空间转录组学配对来了解亚型表达


通过选择性剪接产生的 RNA 亚型多样性，以及转录起始和 poly（A） 位点的替代使用，导致不同的转录本来源于同一基因。不同的亚型可以发挥重要的生物学作用，包括通过改变蛋白质产物的序列或表达水平。最初的 RNA 测序单细胞方法和后来的空间方法现在广泛用于鉴定差异表达基因，它们依赖于短读长，并提供了转录组学比较不同细胞类型的能力，但它们测量差异亚型表达的能力有限。最近，长读长测序方法已与单细胞和空间技术相结合，以表征亚型表达。在这篇综述中，我们概述了单细胞和空间长读长测序的出现，并讨论了与实施这些技术和解释这些数据相关的挑战。我们讨论了它们为理解转录分子的不同可变元件之间的关系提供的机会，并重点介绍了它们被用来表征亚型在发育和病理学中的作用的一些方式。还讨论了单核长读长测序，这是单细胞方法的一个特殊情况。我们试图涵盖这些技术的局限性及其在扩展我们对 RNA 亚型生物学作用仍然有限的理解方面的巨大潜力。