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.