Low-cost high-throughput targeted sequencing for the accurate detection of respiratory tract pathogens

Introduction: The current gold standard for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) diagnosis by real-time reverse transcriptase polymerase chain reaction (RT-PCR) is limited by the number of genes that can be detected. In this study, we developed a lowcost and high-throughput next-generation sequencing technology that can overcome the limitations of real time RT-PCR. Methodology: A targeted sequencing panel (TSP) consisting of approximately 500 amplicons was designed. This panel could simultaneously detect a broad range of gene loci of SARS-CoV-2, and genes for the most common infectious viruses that affect the respiratory tract, in a single run and could include up to 96 samples. Four hundred and forty-eight samples and 31 control samples were analyzed independently with both TSP and RT-PCR, and the results were compared for accuracy and other indicators. Results: TSP identified 50 SARS-CoV-2 positive samples with a 99.33% match to RT-PCR results. It is not surprising that TSP also identified multiple infections from the 96 samples, whereas RT-PCR could not. Thus, TSP was able to accurately diagnose the samples which could not be identified based on single RT-PCR test. Conclusions: Our data demonstrated that TSP is a fast and accurate testing method for identifying multiple pathogen infections of the respiratory tract.