Rapid High-Throughput Whole-Genome Sequencing of SARS-CoV-2 by Using One-Step Reverse Transcription-PCR Amplification with an Integrated Microfluidic System and Next-Generation Sequencing

The long-lasting global COVID-19 pandemic demands timely genomic investigation of SARS-CoV-2 viruses. Here, we report a simple and efficient workflow for whole-genome sequencing utilizing one-step reverse transcription-PCR (RT-PCR) amplification on a microfluidic platform, followed by MiSeq amplicon sequencing. The method uses Fluidigm integrated fluidic circuit (IFC) and instruments to amplify 48 samples with 39 pairs of primers, including 35 custom-designed primer pairs and four additional primer pairs from the ARTIC network protocol v3. Application of this method on RNA samples from both viral isolates and clinical specimens demonstrates robustness and efficiency in obtaining the full genome sequence of SARS-CoV-2.