A sensitive sample preparation pipeline for adventitious virus detection using Oxford Nanopore sequencing.

Area of Science:

• Biotechnology
• Genomics
• Virology

Background:

• Regulatory bodies now favor sequencing over traditional *in vivo* assays for adventitious agent testing in cell line qualification.
• Current short-read sequencing methods for adventitious agent detection are time-consuming, often exceeding a week for results, hindering timely release of critical cell and gene therapies.
• Oxford Nanopore sequencing offers real-time analysis, potentially reducing assay duration, but host nucleic acid background can obscure low-level viral signals.

Purpose of the Study:

• To develop a sensitive and rapid sample preparation workflow for detecting viral contaminants in cell and gene therapy manufacturing using Oxford Nanopore sequencing.
• To overcome the challenge of high background nucleic acid from host cells that can mask low-level viral contaminants.
• To achieve a significant improvement in the limit of detection for viral contaminants, comparable or superior to existing short-read methods.

Main Methods:

• Development of a novel sample preparation workflow involving nucleic acid concentration and nuclease treatment to reduce host background.
• Utilizing agnostic PCR methods to amplify potential viral contaminant sequences.
• Employing Oxford Nanopore sequencing for real-time data acquisition and analysis.

Main Results:

• The developed workflow achieved a 3-log improvement in the limit of detection for viral contaminants.
• The enhanced sensitivity is comparable to or better than current short-read sequencing approaches.
• The method effectively reduces background signals from host nucleic acids, allowing for clearer detection of low-level viral contaminants.

Conclusions:

• The novel sample preparation workflow combined with Oxford Nanopore sequencing provides a rapid and sensitive method for adventitious viral contaminant detection.
• This approach significantly enhances the limit of detection, addressing a critical need in cell and gene therapy manufacturing.
• The findings support the adoption of faster, more sensitive sequencing-based assays for ensuring the safety of cell and gene therapies.