TERA-Seq: true end-to-end sequencing of native RNA molecules for transcriptome characterization

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Direct nanopore sequencing of native RNA offers transformative potential for RNA biology and diagnostics.
• Current nanopore platforms have limitations in sequencing RNA 5' ends and are restricted to polyadenylated molecules.

Purpose of the Study:

• To develop a novel platform, True End-to-end RNA Sequencing (TERA-Seq), to overcome existing limitations in direct RNA sequencing.
• To enable thorough transcriptome characterization, including non-polyadenylated RNAs and precise 5'/3' end identification.

Main Methods:

• TERA-Seq utilizes uniquely designed adapters ligated to RNA molecules for sequencing.
• The platform sequences both polyadenylated and non-polyadenylated RNA, capturing native 5' and 3' ends.

Main Results:

• TERA-Seq identified significant variation in poly(A) tail lengths of capped, full-length mRNAs at the single-molecule level.
• Prevalent non-canonical capping downstream of transcriptional start sites was observed.
• The study revealed cotranslational mRNA decay, often initiated from the 5' end, with retained poly(A) tails.

Conclusions:

• TERA-Seq provides a comprehensive approach for end-to-end direct RNA sequencing, enhancing transcriptome analysis.
• The platform is valuable for applications requiring precise characterization of RNA isoforms and their modifications.
• Findings offer new insights into RNA processing, decay dynamics, and regulatory mechanisms.