Direct RNA sequencing in plants: Practical applications and future perspectives

• Published in: Plant communications 2024-11, Vol.5 (11), p.101064, Article 101064
• Main Authors: Zhu, Xi-Tong, Sanz-Jimenez, Pablo, Ning, Xiao-Tong, Tahir ul Qamar, Muhammad, Chen, Ling-Ling
• Format: Article
• Language: English
• Subjects: direct RNA sequencing; fusion transcripts; High-Throughput Nucleotide Sequencing - methods; novel isoforms; Plants - genetics; poly(A) tail; Review; RNA modifications; RNA, Plant - genetics; Sequence Analysis, RNA - methods; Transcriptome
• ISSN: 2590-3462; 2590-3462
• DOI: 10.1016/j.xplc.2024.101064

The transcriptome serves as a bridge that links genomic variation to phenotypic diversity. A vast number of studies using next-generation RNA sequencing (RNA-seq) over the last 2 decades have emphasized the essential roles of the plant transcriptome in response to developmental and environmental conditions, providing numerous insights into the dynamic changes, evolutionary traces, and elaborate regulation of the plant transcriptome. With substantial improvement in accuracy and throughput, direct RNA sequencing (DRS) has emerged as a new and powerful sequencing platform for precise detection of native and full-length transcripts, overcoming many limitations such as read length and PCR bias that are inherent to short-read RNA-seq. Here, we review recent advances in dissecting the complexity and diversity of plant transcriptomes using DRS as the main technological approach, covering many aspects of RNA metabolism, including novel isoforms, poly(A) tails, and RNA modification, and we propose a comprehensive workflow for processing of plant DRS data. Many challenges to the application of DRS in plants, such as the need for machine learning tools tailored to plant transcriptomes, remain to be overcome, and together we outline future biological questions that can be addressed by DRS, such as allele-specific RNA modification. This technology provides convenient support on which the connection of distinct RNA features is tightly built, sustainably refining our understanding of the biological functions of the plant transcriptome. Direct RNA sequencing (DRS) is revolutionizing plant transcriptome research by enabling the precise characterization of full-length transcripts and their modifications. This review summarizes recent DRS applications in plant research, highlighting their potential to unravel the complexities of plant transcriptomes, while also identifying key challenges and future perspectives in this emerging field.