| Summary: | Transcriptome sequencing revolutionized the analysis of gene expression, providing an unbiased approach to gene detection and quantification that enabled the discovery of novel isoforms, alternative splicing events and fusion transcripts. However, although short-read sequencing technologies have surpassed the limited dynamic range of previous technologies such as microarrays, they have limitations, for example, in resolving full-length transcripts and complex isoforms. Over the past 5 years, long-read sequencing technologies have matured considerably, with improvements in instrumentation and analytical methods, enabling their application to RNA sequencing (RNA-seq). Benchmarking studies are beginning to identify the strengths and limitations of long-read RNA-seq, although there remains a need for comprehensive resources to guide newcomers through the intricacies of this approach. In this Review, we provide a comprehensive overview of the long-read RNA-seq workflow, from library preparation and sequencing challenges to core data processing, downstream analyses and emerging developments. We present an extensive inventory of experimental and analytical methods and discuss current challenges and prospects.
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