1. Introduction
Direct native RNA sequencing is a novel method for sequencing RNA molecules in their native form without needing to first reverse transcribe them into cDNA. This is made possible by Oxford Nanopore Technologies’ nanopore sequencers which can directly sequence native RNA strands as they pass through a protein nanopore . Unlike traditional sequencing methods, direct RNA sequencing can identify RNA modifications, which are typically erased by widely used sequencing-by-synthesis (SBS) methods . This method has been used to document nucleotide modifications and 3′ polyadenosine tails on RNA strands without added chemistry steps . Direct RNA sequencing allows for the analysis of native RNA strands without reverse transcription or amplification, avoiding biases introduced by these steps (Vacca et al. 2022, Soneson et al. 2019).
Over the past few years, direct RNA sequencing accuracy and throughput have improved to the point that it can offer valuable biological insights. For example, it has revealed capping patterns in human mRNAs , detected novel pseudouridine sites in yeast , and quantified changing modification levels under stress . As the technology continues advancing, direct sequencing of full-length native RNA strands promises to transform transcriptomics.
Direct RNA sequencing has some limitations to consider. Current protocols require high-quality input RNA, with recommendations of at least 50ng of intact mRNA for optimal throughput . This high RNA input requirement could pose challenges for studies with limited biological material . Additionally, the protocols rely on the poly(A) tail for adapter ligation, restricting the analysis to polyadenylated transcripts and limiting the characterization of non-polyadenylated RNAs . The throughput of direct RNA sequencing is also currently lower than short-read methods based on cDNA sequencing. This can restrict the depth of characterization possible for complex transcriptomes . Finally, computational tools tailored for analyzing the direct sequencing data are still in early development, making data analysis more difficult than established pipelines for short-read data . Further advances in methods and tools will help address these current limitations of direct RNA sequencing, including increased output and error reduction in incoming RNA004 kits.
While direct RNA sequencing has some limitations, it also presents exciting opportunities to advance transcriptome profiling, especially in non-model organisms exhibiting remarkable environmental adaptability like amphibious plants that exhibit remarkable adaptability, adjusting their morphology and physiology to thrive in fluctuating aquatic and terrestrial environments. Recent advances in genomics and transcriptomics have shed light on the genetic mechanisms underlying aquatic adaptation. Comparative transcriptomics of amphibious plants grown submerged versus on land reveal differentially expressed genes involved in underwater acclimation like cuticle and stomatal development, cell elongation, and modified photosynthesis . Genomics has also uncovered key roles of plant hormones in regulating heterophylly . Moreover, comparative genomics between aquatic and terrestrial species identify genomic signatures enabling adaptation to submerged life, including changes in submergence tolerance, light sensing, and carbon assimilation genes . However, genomic resources for amphibious plants remain scarce especially in the non-vascular evolutionary lineage.
Riccia fluitans is an aquatic liverwort that serves as an excellent model for studying amphibious plants. As one of the earliest diverging land plants, liverworts represent a critical transition point between aquatic and terrestrial environments . R. fluitanspossess remarkable adaptability, growing floating mats in water or moist soil . When submerged, R. fluitans adopts a specialized water form with thin thalli to maximize surface area for gas exchange. Within days of emerging from the water, it can completely alter its morphology into a land form with thicker thallus that reduces water loss. It also stockpiles starch preparing for periodic drought . This extreme plasticity enables the exploitation of both aquatic and terrestrial realms. Its ability to dynamically transform morphology and physiology demonstrates exceptional environmental responsiveness. Elucidating the adaptations underlying such plasticity provides perspective on water-to-land transitions of early land plants over 400 million years ago . As an amphibious plant that flourishes both submerged and on moist land, R. fluitans serves as a prime model for examining adaptive mechanisms to alternating hydrological regimes. The recent establishment of genetic transformation methods unlocks additional potential for exploring the genetic basis of aquatic acclimation in this liverwort .
In this study, we analyze land and water forms of Riccia fluitansusing nanopore native RNA sequencing technology to verify if this technology could provide additional insight into short-read characterized transcriptomes as well as potential epitranscriptomics changes during adaptation to aquatic environments, which wasn’t studied in liverworts so far.