Long-Read Sequencing for mRNA Vaccine Characterization

At a glance:

• Introduction
• What Is Long-Read Sequencing for mRNA Vaccine Characterization?
• Key Advantages of Our mRNA Vaccine Characterization Service
• Applications of Long-Read Sequencing for mRNA Vaccine Characterization
• Technology Overview – How We Characterize mRNA Vaccines
• Bioinformatics Analysis
• Choosing the Right Method for mRNA Vaccine QC
• Sample Requirements
• Why Choose CD Genomics
• Case Study: mRNA Vaccine Quality Analysis Using Long-Read RNA Sequencing
• FAQs
• Demo

What Is Long-Read Sequencing for mRNA Vaccine Characterization?

Long-read sequencing for mRNA vaccine characterization uses full-length cDNA sequencing and direct native RNA sequencing to read an in-vitro transcribed mRNA molecule end to end, without breaking it into short fragments first. Because each read spans the complete transcript, the resulting data captures sequence identity, structural integrity, and 3′ poly(A) tail length simultaneously, and direct RNA sequencing further preserves chemical modifications such as N1-methylpseudouridine that are normally lost or averaged out by reverse transcription.

This capability matters because a single truncation event, sequence variant, or contaminating species anywhere along an mRNA can compromise a batch, and fragment-based methods are poorly suited to localizing exactly where and how often these defects occur. Long-read data lets our bioinformatics team map defects to specific positions in the transcript, quantify their frequency across the read population, and distinguish the intact drug substance from partial or off-target byproducts.

The approach is equally relevant to earlier-stage work on the DNA template. Our full-length plasmid sequencing service can confirm the linearized production plasmid or PCR template before in-vitro transcription, so that identity issues are caught upstream rather than discovered only after mRNA synthesis.

Applications of Long-Read Sequencing for mRNA Vaccine Characterization

Sequence Identity and Integrity Confirmation

• Confirm that the manufactured mRNA matches the intended design, including the 5′ UTR, open reading frame, and 3′ UTR.
• Detect and localize truncated or prematurely terminated transcripts.
• Identify sequence variants introduced during template construction or in-vitro transcription.

Poly(A) Tail and Untranslated Region Analysis

• Measure poly(A) tail length distribution directly on full-length reads.
• Assess UTR sequence consistency across a transcript population.
• Support formulation and stability studies that depend on tail length uniformity.

Modified Nucleoside Verification

• Confirm incorporation of modified nucleosides such as N1-methylpseudouridine using direct RNA sequencing.
• Screen for incomplete or inconsistent modification across the transcript.
• Support chemistry comparisons between candidate mRNA constructs, extending the same principles used in our broader long-read RNA methylation analysis work.

Contaminant and Byproduct Screening

• Detect residual linearized plasmid, run-off transcripts, and double-stranded RNA byproducts of in-vitro transcription.
• Quantify off-target RNA species alongside the intended mRNA drug substance.
• Support comparability studies between manufacturing batches or process changes.

Platform and Construct Comparison

• Compare candidate UTR, cap, or poly(A) designs for their effect on integrity and modification profile.
• Benchmark new formulations against reference standards for non-clinical research programs.

Bioinformatics Analysis

Sample Requirements

Case Study: mRNA Vaccine Quality Analysis Using Long-Read RNA Sequencing

Gunter, H.M., Idrisoglu, S., Singh, S. et al. mRNA vaccine quality analysis using RNA sequencing. Nature Communications 14, 5663 (2023).

1. Background

mRNA vaccines must be rigorously analysed to measure their integrity and detect contaminants that can reduce effectiveness or induce side effects, yet the industry-standard toolkit of RT-qPCR, chromatography, and capillary or gel electrophoresis is time-consuming and cannot resolve every quality attribute in one assay. This study, from the BASE facility at the University of Queensland, set out to test whether long-read nanopore sequencing could measure key mRNA vaccine quality attributes in a single, streamlined workflow, an approach the authors termed VAX-seq.

2. Methods

The authors validated VAX-seq using a reference eGFP mRNA construct comprising a 5′ T7 CleanCap promoter, an alpha-globin 5′ UTR, an eGFP open reading frame, an AES/mtRNR1 3′ UTR, and a 126 nt poly(A) tail, cloned into a pUC-57 plasmid backbone and linearized prior to in-vitro transcription. Following purification to remove residual DNA, RNA, and protein, the mRNA was analysed by long-read cDNA sequencing on the Oxford Nanopore platform, with reads processed through the authors' supporting Mana software to determine sequence identity, transcript length, and integrity, and to measure poly(A) tail length. To evaluate mRNA chemistry, the team separately generated mRNA incorporating N1-methylpseudouridine using CleanCap AG capping and modified nucleotide reagents, then applied direct RNA sequencing to detect the incorporated modification without reverse transcription.

3. Results

Long-read cDNA sequencing confirmed the sequence identity, length, and integrity of the reference eGFP mRNA and provided a poly(A) tail length read-out in the same workflow used to assess sequence quality, alongside detection of off-target RNA contaminants that can arise during in-vitro transcription. For the chemistry arm of the study, direct RNA sequencing of the N1-methylpseudouridine-containing mRNA successfully detected the modified nucleoside in single mRNA molecules, although the authors reported that direct RNA sequencing libraries yielded lower output and a higher error rate than matched cDNA sequencing libraries for the same construct.

4. Conclusions

The study demonstrates that long-read nanopore sequencing can serve as a single, streamlined method for characterizing mRNA vaccines and therapeutics. Importantly:

• Sequence identity, length, and integrity can be confirmed from one long-read cDNA sequencing workflow
• Poly(A) tail length is captured directly, alongside sequence-level quality metrics
• Off-target RNA contaminants introduced during manufacturing can be detected
• Direct RNA sequencing can detect incorporated modified nucleosides such as N1-methylpseudouridine in native mRNA
• The authors anticipate that RNA sequencing methods such as VAX-seq will become central to mRNA vaccine and therapeutic development

Demo

1. Full-Length Read Coverage and Truncation Map

2. Poly(A) Tail Length Distribution

3. Modified Nucleoside Detection Signal (Direct RNA Sequencing)