Serge Monpoeho from Regeneron Pharmaceuticals in the US presented at the ONT Biopharma Day in Boston. The title of the session was “Adventitious agent screening by long-read NGS.” They wanted to develop an Oxford Nanopore-driven adventitious agent detection system. They used the Ligation Sequencing Kit, heat extraction, and custom universal PCR primers. They ran for 48 hours. The bioinformatics analysis consisted of generating FASTQs, quality control, and filtering, read alignment to a genome database, and then to a protein database. They used criteria to define hits based on coverage. They used the FDA AVDTIG sample for spiking. One set of samples was additionally spiked with MS1 phage. Monpoeho shared results of which viruses were detected and at what levels. They were able to detect most spiked in viruses in their samples but also their negative (water) control. They attributed this to reagent and adenovirus background contamination. Monpoeho explained that the challenges include assay validation, which virus models to use, and Q-line instrument/reagents. Database updates must also follow a regulated process along with the computer/cloud system pipeline validation. Speed and cost analyses are necessary to determine the impact this testing process will have on the cost of the product. Monpoeho spoke about improving the purification process to reduce host background and improving the pre-amplification… or eliminating this step. The controls used for the assay will also have to be carefully considered. Monpoeho explained that the Q-score for the run and negative control should be considered. The unmapped reads could be further characterized, too. I learned that even with sensitive detection methods, this type of assay will be challenging to design and validate!
