Integrating multiplex digital PCR and long-read sequencing toward standardized genome integrity and purity characterization of rAAV9.
4 affiliations
- 1School of Natural Sciences, University of Kent, Canterbury, Kent CT2 7NJ, UK.
- 2National Measurement Laboratory at LGC, Guildford, Surrey GU2 7XY, UK.
- 3Reading Scientific Services Ltd., Reading Science Centre, Whiteknights Campus, Pepper Lane, Reading, Berkshire RG6 6LA, UK.
- 4National Institute for Bioprocessing Research and Training (NIBRT), Foster Avenue, Mount Merrion, Blackrock, Co. Dublin A94 X099, Ireland.
Abstract
Recombinant adeno-associated virus (rAAV) production can yield heterogeneous viral particle populations containing incomplete genomes and co-packaged DNA impurities that may compromise dosing accuracy and safety. Here, we applied long-read next-generation sequencing (NGS) to characterize the genome and DNA impurity profiles of rAAV9 and compare genome integrity estimates with digital PCR (dPCR). A 4-plex dPCR assay targeting four regions of the rAAV9 genome was developed to enable simultaneous assessment of genome titer and integrity in both intact rAAV9 particles and extracted genomes. To prepare samples for both dPCR and long-read NGS, rAAV9 genomes were released using a heat-based protocol or a commercial extraction kit and sequenced. Long-read NGS revealed the presence of host cell DNA, plasmid DNA, and chimeric reads. The chimeric reads spanned from the inverted terminal repeats sequences into the transfer plasmid backbone, potentially due to mispackaging events. Genome integrity estimates differed significantly between methods, with dPCR reporting higher genome integrity than long-read NGS. Together, multiplex dPCR and long-read NGS provide complementary information on rAAV critical quality attributes and support more comprehensive quality control of rAAV batches across process development and batch release.
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BibTeX
@article{kontogiannis2026integrating,
title = {{Integrating multiplex digital PCR and long-read sequencing toward standardized genome integrity and purity characterization of rAAV9.}},
author = {Theodoros Kontogiannis and Rui P A Pereira and Carole Foy and Julian Braybrook and Christopher McElroy and Milena Quaglia and C Mark Smales and Alexandra S Whale},
journal = {Molecular therapy. Advances},
year = {2026},
month = sep,
doi = {10.1016/j.omta.2026.201851},
url = {https://europepmc.org/article/MED/42909404},
note = {CC BY-NC-ND},
}
RIS
TY - JOUR
TI - Integrating multiplex digital PCR and long-read sequencing toward standardized genome integrity and purity characterization of rAAV9.
AU - Theodoros Kontogiannis
AU - Rui P A Pereira
AU - Carole Foy
AU - Julian Braybrook
AU - Christopher McElroy
AU - Milena Quaglia
AU - C Mark Smales
AU - Alexandra S Whale
PY - 2026
DA - 2026/09/28
JO - Molecular therapy. Advances
DO - 10.1016/j.omta.2026.201851
UR - https://europepmc.org/article/MED/42909404
AB - Recombinant adeno-associated virus (rAAV) production can yield heterogeneous viral particle populations containing incomplete genomes and co-packaged DNA impurities that may compromise dosing accuracy and safety. Here, we applied long-read next-generation sequencing (NGS) to characterize the genome and DNA impurity profiles of rAAV9 and compare genome integrity estimates with digital PCR (dPCR). A 4-plex dPCR assay targeting four regions of the rAAV9 genome was developed to enable simultaneous assessment of genome titer and integrity in both intact rAAV9 particles and extracted genomes. To prepare samples for both dPCR and long-read NGS, rAAV9 genomes were released using a heat-based protocol or a commercial extraction kit and sequenced. Long-read NGS revealed the presence of host cell DNA, plasmid DNA, and chimeric reads. The chimeric reads spanned from the inverted terminal repeats sequences into the transfer plasmid backbone, potentially due to mispackaging events. Genome integrity estimates differed significantly between methods, with dPCR reporting higher genome integrity than long-read NGS. Together, multiplex dPCR and long-read NGS provide complementary information on rAAV critical quality attributes and support more comprehensive quality control of rAAV batches across process development and batch release.
SN - 3117-387X
ER -