Direct RNA sequencing by nanopore: a breakthrough in RNA vaccine quality control with cost reduction and high accuracy

Introduction: RNA-based vaccines have revolutionized immunization by enabling rapid responses to emerging diseases.Ensuring RNA integrity, including poly(A) tail assessment, is critical for stability and efficient antigen expression.Traditional quality control methods-mass spectrometry, capillary electrophoresis, and NGS-are effective but costly, time-consuming, and require sophisticated infrastructure, with some limitations in accuracy.Direct RNA sequencing by Nanopore offers a costeffective, rapid alternative.Objectives: This study aimed to evaluate Nanopore sequencing for RNA vaccine quality control, focusing on poly(A) tail length and sequence identity, which are vital for stability and translation efficiency.Methodology: RNA was synthesized in vitro (IVT), and integrity was assessed by capillary electrophoresis, achieving >90% integrity.Sequencing was performed using the Nanopore Direct RNA sequencing kit, enabling real-time analysis while preserving the native RNA strand, despite reverse transcription.Data analysis was conducted using DORADO for basecalling and poly(A) tail assessment, and Geneious for sequence identity verification.Results: 15% of variation in poly(A) length was observed.Despite this variation, experimental results remained robust and aligned with performance parameters, demonstrating the reliability of our manufacturing process.Unlike mass spectrometry, which estimates poly(A) length based on fragment mass, Nanopore sequencing directly analyzes individual RNA molecules, providing a distribution of poly(A) tail lengths across reads.This enables comprehensive characterization and supports process refinement for enhanced quality control.Data quality was excellent, with mean Q-scores above Q20, ensuring an error rate below 1%.While NGS platforms achieve Q30, Nanopore's direct RNA sequencing eliminates biases from reverse transcription, making it significantly more reliable than traditional methods that accept a minimum Q10.Economic analysis revealed that Nanopore sequencing reduces costs by approximately 70% compared to mass spectrometry, factoring in equipment installation and environmental adaptations.It is also 40% more cost-effective than NGS, with an estimated cost of ~US$ 500 per sample. Conclusion:These findings position Nanopore sequencing as a transformative tool for RNA vaccine quality control, providing real-time, high-accuracy insights into sequence integrity.Its ability to streamline industrial and regulatory workflows accelerates decision-making and enhances efficiency, representing a shift in biotechnology and advancing RNA vaccine development.