Webinar on Thursday, June 24, 2021
8:00 am PT | 11:00 am ET | 4:00 pm BST | 5:00 pm CEST
Tao Li, MS
Viral Diseases Branch
Center of Infectious Diseases Research
Walter Reed Army Institute of Research
The global COVID-19 pandemic demands timely genomic investigation of SARS-CoV-2 viruses. Genomic surveillance has become a critical measure for identification of new SARS-CoV-2 variants, providing vital information for infection control strategies and vaccine development. In contrast to the massive capacity of next-generation sequencing (NGS), conventional methods for whole genome amplification of RNA viruses are often time-consuming, a big hurdle for amplification of large 30 Kb genomic RNA such as that of SARS-CoV-2. Using the Fluidigm integrated fluidic circuit (IFC) and Access Array™ instruments, we built a robust workflow for sequencing, utilizing one-step whole-genome reverse transcription PCR (RT-PCR) amplification, followed by amplicon sequencing using Illumina® NGS systems MiSeq™, NextSeq™ or NovaSeq™. The 48.48 Access Array™ IFC was used to amplify 48 extracted RNA samples with up to 48 individual pairs of primers. Only 1 to 1.45 µL of RNA was needed for each sample with minimal hands-on time and lower risks of contamination. The yields of RT-PCR and genome assembly coverage were highly correlated with Ct values or viral titers of the samples. The method has been successfully used in a number of projects on RNA samples from both viral isolates and clinical specimens, with demonstrated robustness and efficiency in obtaining the full genome sequence of SARS-CoV-2.
For Research Use Only. Not for use in diagnostic procedures.
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