Alternative splicing (AS) stands as a foundational biological process, allowing a single gene to produce multiple RNA isoforms. AS plays a pivotal role in the human brain giving rise to a myriad of isoforms characterized by variations in structure, function, and expression patterns. These RNA isoforms hold significance due to their influence on cell states, functions, and their regulatory roles in crucial neurodevelopmental processes and disease pathways. However, our understanding of RNA isoforms and which cell-types they are expressed in has been circumscribed by the limitations of short-read sequencing methods. With the advent of long-read sequencing technologies, such as Nanopore sequencing, a comprehensive examination of the isoform landscape is now possible, providing the opportunity to explore isoforms expression dynamics, isoform structure and their intricate contributions to both developmental processes and disease pathogenesis.
In this context, we have devised an innovative long-read-only single-cell sequencing methodology that provides an unparalleled opportunity to explore isoforms at the granularity of individual cells. With this approach, we explore neurogenesis in differentiating cortical excitatory projection neurons and cerebral organoids. Our findings showcase that our long-read-only single-cell method has the capacity to delineate distinct cell types, unearth thousands of previously undiscovered isoforms, chart complex developmental trajectories, and pinpoint hundreds of differentially expressed genes and isoforms involved in neurogenesis. We find cases of isoform switching that occurs during cell state transitions and identified alternatively spliced isoforms that impact the function of key glutamatergic receptor subunits. These results showcase the power of our novel method for unravelling isoforms associated with both development and disease and paves the way for a promising exploration of the expansive isoform landscape and the underlying mechanisms that shape complex developmental systems such as the brain.