New targeted next-generation sequencing (NGS) panel for detection of acute respiratory infection pathogens

Respiratory infections are among the leading causes of morbidity and mortality worldwide, especially among children, the elderly, and immunocompromised individuals. To address the limitations of the existing diagnostic tools and improve testing efficiency, we developed, optimized and tested in-house targeted next-generation sequencing assay capable of simultaneously detecting 32 groups of viruses and 13 bacteria commonly associated with respiratory infections. Using NGS-PrimerPlex, we designed a total of 117 primer pairs-at least two per pathogen-targeting different genomic regions. These primers were divided into three multiplex PCR reactions and evaluated both in silico for specificity and coverage, and in vitro via monoplex PCR with positive controls and polyacrylamide gel electrophoresis. We systematically optimized key variables including primer (15-260 nM) and dNTP (0.1-1.6 mM) concentrations, PCR cycle number (20-35), annealing temperature (57-62°C), ramp rate (0.2-4°C/s), and reverse transcription protocols. The assay was validated using 120 complementary DNA (cDNA) samples from patients at Speransky Children's Hospital #9 in Moscow, Russia, previously tested with a commercial multiplex PCR respiratory virus panel. The NGS assay showed complete concordance with the PCR results in 60% of positive samples, increasing to 74% when samples with Cq values above 29 were excluded. Additionally, the NGS assay detected target pathogens in 13 PCR-negative samples and identified other respiratory pathogens not included in the commercial panel in 47 PCR-negative samples. Overall, the assay identified at least one target pathogen in 86 of 120 samples, demonstrating its potential for comprehensive respiratory pathogen detection, surveillance, and clinical diagnostics. Our findings offer a robust foundation for future development of targeted NGS-based assays.