Direct RNA sequencing in plants: practical applications and future perspectives.

The transcriptome serves as a bridge that links genomic variation and phenotype diversity. A vast number of studies using next-generation RNA sequencing (RNA-seq) in the last two decades emphasize the essential roles of plant transcriptome in response to developmental and environmental conditions, leading to numerous insights into the dynamic change, evolutionary trace and elaborate regulation of plant transcriptome. With substantial improvement in accuracy and throughput, direct RNA sequencing (DRS) has emerged as a new and powerful sequencing platform for the precise detection of native and full-length transcripts, which overcomes many limitations such as read length and PCR bias that are inherent to short-read RNA-seq. Here, we reviewed recent advances in dissecting the complexity and diversity of plant transcriptome utilizing DRS as a main technological mean from many aspects of RNA metabolism, including novel isoforms, poly(A) tail and RNA modification, and proposed a comprehensive workflow for the data process of plants DRS. Many challenges concerning the application of DRS in plants, such as machine learning tools tailored to plant transcriptome, remain to be solved, and together we prospect the future biological questions that can be potentially answered 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 plant transcriptome.