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Direct full-length RNA sequencing reveals unexpected transcriptome complexity during Caenorhabditis elegans
Runsheng Li1, Xiaoliang Ren1, Qiutao Ding1
1Department of Biology, Hong Kong Baptist University, Hong Kong, 999077, China.
Genome Research
|February 7, 2020
Summary
Direct RNA sequencing with ultralong reads reveals novel RNA isoforms and modifications in *C. elegans*. This advanced method enhances understanding of transcriptome complexity and RNA processing across developmental stages.
Area of Science:
- Genomics
- Molecular Biology
- Bioinformatics
Background:
- RNA sequencing (RNA-seq) is crucial for understanding transcriptome complexity, including alternative splicing and RNA modifications.
- Current short-read RNA-seq technologies limit the comprehensive analysis of full-length transcripts and RNA modifications.
- Direct RNA sequencing offers a promising alternative for capturing long, native RNA molecules.
Purpose of the Study:
- To investigate transcriptome complexity in *Caenorhabditis elegans* using direct RNA sequencing with ultralong reads.
- To develop a novel method for classifying long RNA reads to identify novel and existing transcript isoforms.
- To explore the landscape of RNA modifications within coding and untranslated regions.
Main Methods:
- Application of Oxford Nanopore Technologies for direct RNA sequencing of poly(A)-tailed mRNAs from three *C. elegans* developmental stages.
- Generation of approximately six million ultralong reads with average lengths of 900–1100 nt, with about half representing full-length transcripts.
- Development of a sequence mapping-based read classification method to identify novel transcript isoforms, distinct from traditional intron/exon structure analysis.
Main Results:
- Identification of approximately 57,000 novel transcript isoforms and recovery of at least 26,000 existing isoforms.
- Observation of distinct gene sets showing differential expression versus differential isoform usage during development, indicating fine-tuned isoform-level regulation.
- Detection of an increased prevalence of putative RNA modifications in coding regions compared to untranslated regions (UTRs).
Conclusions:
- Direct RNA sequencing with ultralong reads significantly enhances the ability to define transcriptome complexity and identify novel RNA isoforms.
- Isoform-level regulation plays a critical role in developmental processes, complementing gene expression changes.
- The observed RNA modifications in coding regions may have functional implications for translation, warranting further investigation.
