An Iterative Approach to Polish the Nanopore Sequencing Basecalling for Therapeutic RNA Quality Control

Area of Science:

• Molecular Biology
• Bioinformatics
• Genomics

Background:

• Nanopore sequencing is a powerful tool for direct nucleic acid analysis.
• Nucleotide modifications introduce artifacts in nanopore sequencing, compromising basecalling accuracy.
• Accurate basecalling is essential for analyzing modified nucleic acids, including therapeutic RNAs.

Purpose of the Study:

• To develop and validate an iterative approach for polishing basecalling results affected by nucleotide modifications.
• To enhance the accuracy of nanopore sequencing for both synthetic and native RNA molecules.
• To apply the improved basecalling method for critical applications in therapeutic RNA analysis.

Main Methods:

• Development of an iterative algorithm to correct basecalling errors caused by nucleotide modifications.
• Testing the approach on artificially synthesized and real-world RNA samples.
• Application of the method to analyze vaccine messenger RNAs (mRNAs) and RNA interference (RNAi) molecules.

Main Results:

• The iterative approach significantly improves basecalling accuracy in the presence of nucleotide modifications.
• Demonstrated efficacy on both synthesized and naturally occurring RNA molecules.
• Successful application for purity and integrity assessment of vaccine mRNAs and identification of modification sites in novel RNAi therapeutics.

Conclusions:

• The developed iterative method effectively corrects nanopore sequencing artifacts from nucleotide modifications.
• This approach provides a reliable basis for precise basecalling of therapeutic RNAs.
• Enables accurate quantification of vaccine mRNA quality and characterization of novel RNA-based therapeutics.