Improved long-transcript representation in Oxford Nanopore direct RNA sequencing with UltraMarathonRT

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Direct RNA sequencing (DRS) enables native RNA analysis, bypassing PCR amplification biases.
• Current DRS protocols use high-temperature reverse transcriptase (RT) that can degrade RNA.
• Oxford Nanopore (ONT) sequencing is a key technology for DRS.

Purpose of the Study:

• To develop an improved DRS protocol for longer RNA reads and more accurate isoform prediction.
• To evaluate the performance of a novel reverse transcriptase in DRS library preparation.
• To address RNA degradation issues in existing DRS methods.

Main Methods:

• Incorporation of UltraMarathonRT (uMRT), an ultraprocessive RT with helicase activity optimal at 30°C.
• Optimization of library preparation steps for ONT DRS.
• Testing the new protocol on Universal Human Reference RNA and human brain RNA samples.

Main Results:

• The uMRT-based DRS method yields significantly longer RNA reads compared to standard protocols.
• Improved RNA integrity and reduced degradation were observed.
• More accurate and longer final isoform predictions were achieved.

Conclusions:

• The novel uMRT-based DRS protocol enhances RNA sequencing accuracy and read length.
• This improved workflow minimizes RNA degradation, enabling more comprehensive native RNA analysis.
• The method has the potential to drive new discoveries in RNA biology and genomics.