Highly sensitive and full-genome interrogation of SARS-CoV-2 using multiplexed PCR enrichment followed by next-generation sequencing

Many detection methods have been used or reported for the diagnosis and/or surveillance of COVID-19 Amongthem, reverse transcription polymerase chain reaction (RT-PCR) is the most commonly used because of its highsensitivity, typically claiming detection of about 5 copies of viruses However, it has been reported that only 47-59%of the positive cases were identified by some RT-PCR methods, probably due to low viral load, timing of sampling, degradation of virus RNA in the sampling process, or possible mutations spanning the primer binding sites Therefore, alternative and highly sensitive methods are imperative With the goal of improving sensitivity andaccommodating various application settings, we developed a multiplex-PCR-based method comprising 343 pairs ofspecific primers and demonstrated its efficiency at detecting SARS-CoV-2 at low copy numbers The assayproduced clean characteristic target peaks of defined sizes, which allowed for direct identification of positives byelectrophoresis We further amplified the entire SARS-CoV-2 genome from 8 to half a million viral copies purifiedfrom 13 COVID-19 positive specimens and detected mutations through next-generation sequencing Finally, wedeveloped a multiplex-PCR-based metagenomic method in parallel that required modest sequencing depth foruncovering SARS-CoV-2 mutational diversity and potentially novel or emerging isolates