In recent years, third-generation sequencing (TGS) technologies have transformed genomics and transcriptomics research, providing novel opportunities for significant discoveries. The long-read sequencing platforms, with their unique advantages over next-generation sequencing (NGS), including a definitive protocol, reduced operational time, and real-time sequencing, possess the potential to transform plant genomics. TGS optimizes and enhances the efficiency of data analysis by removing the necessity for time-consuming assembly tools. The current review examines the development and application of bioinformatics tools for data analysis and annotation, driven by the rapid advancement of TGS platforms like Oxford Nanopore Technologies and Pacific Biosciences. Transcriptome analysis utilizing TGS has been extensively employed to elucidate complex plant transcriptomes and genomes, particularly those characterized by high frequencies of duplicated genomes and repetitive sequences. As a result, current methodologies that allow for generating transcriptomes and comprehensive whole-genome sequences of complex plant genomes employing tailored hybrid sequencing techniques that integrate NGS and TGS technologies have been emphasized herein. This paper, thus, articulates a vision for a future in which TGS effectively addresses the challenges faced in plant research, offering a comprehensive understanding of its advantages, applications, limitations, and promising prospects.
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