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Rup (RNA-seq Usability Assessment Pipeline) - Quality Control for Bulk RNA-seq Experiments in Eukaryotes

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Assessing mRNA and sgRNA Quality for Cell and Gene Therapy Applications Using Nanopore Direct RNA Sequencing.

Kamalakar Chatla¹, Brian Roper¹, Luladey Ayalew¹

¹Cell Therapy Engineering and Development, Genentech, 1 DNA Way, South San Francisco, California 94080, United States.

Analytical Chemistry

|March 6, 2026

Summary

Nanopore direct RNA sequencing (NDRS) provides a unified platform for characterizing RNA therapeutics. This method accurately assesses critical quality attributes like sequence, integrity, and capping, ensuring RNA drug safety and efficacy.

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Area of Science:

Biotechnology
Molecular Biology
Analytical Chemistry

Background:

RNA therapeutics development requires precise characterization of critical quality attributes (CQAs).
Nanopore direct RNA sequencing (NDRS) offers single-molecule analysis for RNA sequence and structure.
Regulatory expectations necessitate robust analytical methods for RNA CQAs.

Purpose of the Study:

Establish NDRS as a comprehensive, multiattribute analytical platform for RNA therapeutics.
Develop novel strategies to assess key CQAs of mRNA and single-guide RNA (sgRNA) in a single assay.
Validate NDRS performance against established orthogonal techniques.

Main Methods:

Optimized NDRS workflow for full-length mRNA sequencing and integrity assessment.
Developed a sequencing-based method for quantifying 5' capping efficiency.
Incorporated a 5' RNA oligo adapter for full-length sgRNA sequencing.
Performed quantitative analysis of poly(A) tail length and integrity.

Main Results:

NDRS accurately determined full-length mRNA sequences and transcript integrity.
Quantified 5' capping efficiency directly from native RNA molecules.
Achieved full-length sequencing of 100-nucleotide sgRNA molecules for identity verification.
Quantitative results for poly(A) tail length, integrity, and capping efficiency correlated with orthogonal methods.
Demonstrated functional impact of mRNA degradation on CRISPR-Cas9 editing efficiency.

Conclusions:

NDRS is a versatile, unified analytical platform for comprehensive mRNA and sgRNA characterization.
This platform supports quality assurance, comparability, and control in RNA therapeutic development.
NDRS facilitates the manufacturing of next-generation RNA therapeutics.