Abstract S08-01: 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. Among them, reverse transcription polymerase chain reaction (RT-PCR) is the most commonly used because of its high sensitivity, 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 and accommodating various application settings, we developed a multiplex-PCR-based method comprising 343 pairs of specific primers and demonstrated its efficiency at detecting SARS-CoV-2 at low copy numbers. The assay produced clean characteristic target peaks of defined sizes, which allowed for direct identification of positives by electrophoresis. We further amplified the entire SARS-CoV-2 genome from 8 to half a million viral copies purified from 13 COVID-19 positive specimens and detected mutations through next-generation sequencing. Finally, we developed a multiplex-PCR-based metagenomic method in parallel that required modest sequencing depth for uncovering SARS-CoV-2 mutational diversity and potentially novel or emerging isolates. Citation Format: Chenyu Li, David N. Debruyne, Julia Spencer, Vidushi Kapoor, Lily Y. Liu, Bo Zhou, Utsav Pandey, Moiz Bootwalla, Dejerianne Ostrow, Dennis T. Maglinte, David Ruble, Alex Ryutov, Lishuang Shen, Lucie Lee, Rounak Feigelman, Grayson Burdon, Jeffrey Liu, Alejandra Oliva, Adam Borcherding, Hongdong Tan, Alexander E. Urban, Xiaowu Gai, Jennifer Dien Bard, Guoying Liu, Zhitong Liu. Highly sensitive and full-genome interrogation of SARS-CoV-2 using multiplexed PCR enrichment followed by next-generation sequencing [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2020 Jul 20-22. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(18_Suppl):Abstract nr S08-01.