New insights of Arabidopsis transcriptome complexity revealed by direct sequencing of native RNAs

ABSTRACT: The transcriptome profiles of the model plant Arabidopsis thaliana have been extensively studied and charcaterised under different developmental and physiological conditions. However, most of these “RNA-sequencing” datasets have been generated using the sequencing of reverse-transcribed cDNAs from mRNAs that have a relatively short read length. Here, we performed direct RNA sequencing using the latest Oxford Nanopore Technology (ONT) with unusual read length. We demonstrate that the complexity of the A. thaliana transcriptomes has been under-estimated. The ONT direct RNA sequencing technology identified transcript isoforms at a vegetative (14 day old seedlings, stage 1.04) and a reproductive stage (stage 6.00-6) when 10% of the flowers had opened. In-house software called TrackCluster was used to determine alternative transcription initiation (ATI), possible alternative polyadenylation (APA), poly(A) length, alternative splicing (AS), and fusion transcripts. Tombo software was used to detect RNA base modifications. More than 38,500 novel transcript isoforms were identified, including six categories of fusion-transcripts which may result from differential RNA processing mechanisms. Fusion-transcripts are prone to mis-assembly by sequencing with short reads using next-generation-sequencing (NGS). These new transcript isoforms provide important additions to the annotated Arabidopsis genome. The power of ONT in detecting RNA modifications was demonstrated by characterisation of the modifications between mobile mRNAs and total mRNAs. The mobile mRNAs were enriched in m5C modifications, which is consistent with a recent finding that m5C modification in mRNAs is crucial for their long-distance movement. In summary, ONT direct RNA sequencing greatly enhances the identification of novel RNA transcript isoforms and RNA base modifications.

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE144828 | GEO | 2020/08/02

REPOSITORIES: GEO