Defining the True Native Ends of RNAs at Single-Molecule Level with TERA-Seq.

Area of Science:

• Molecular Biology
• Genomics
• Transcriptomics

Background:

• Messenger RNA (mRNA) turnover is crucial for gene expression regulation and removing aberrant transcripts.
• Short-read sequencing methods for mRNA decay profiling miss information from RNA ends, limiting biological insights.
• Oxford Nanopore Technology (ONT) offers direct, single-molecule RNA sequencing but has limitations in 5' end and non-polyadenylated RNA detection.

Purpose of the Study:

• To present a novel method, True End-to-end RNA Sequencing (TERA-Seq), for comprehensive RNA profiling.
• To address the limitations of existing ONT platforms for full-length RNA characterization.
• To enable accurate sequencing of both polyadenylated and non-polyadenylated RNA molecules, capturing native ends.

Main Methods:

• Developed a detailed experimental protocol for TERA-Seq.
• Designed unique adapters for ligation to 5' ends (5TERA), 3' ends (TERA3), or both ends (5TERA3) of RNA molecules.
• Sequenced RNA transcripts along with ligated adapters using ONT.

Main Results:

• TERA-Seq allows accurate representation and characterization of RNAs at the single-molecule level.
• The method successfully sequences both poly- and non-polyadenylated RNA molecules.
• TERA-Seq accurately identifies the native 5' and 3' ends of RNA transcripts.

Conclusions:

• TERA-Seq overcomes current ONT limitations, providing a powerful tool for RNA analysis.
• This method enhances the study of RNA decay mechanisms and regulation by enabling full-length transcript characterization.
• TERA-Seq facilitates a deeper understanding of RNA biology through comprehensive, end-to-end sequencing.