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

Tao Li; Hye Kyung Chung; Papa K Pireku; Brett F Beitzel; Mark A Sanborn; Cynthia Y Tang; Richard D Hammer; Detlef Ritter; Xiu-Feng Wan; Irina Maljkovic Berry; Jun Hang

doi:10.1128/JCM.02784-20

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

J Clin Microbiol. 2021 Apr 20;59(5):e02784-20. doi: 10.1128/JCM.02784-20. Print 2021 Apr 20.

Authors

Tao Li¹, Hye Kyung Chung¹, Papa K Pireku¹, Brett F Beitzel², Mark A Sanborn¹, Cynthia Y Tang^{3

4

5

6}, Richard D Hammer⁷, Detlef Ritter⁷, Xiu-Feng Wan^{3

4

5

6

8}, Irina Maljkovic Berry¹, Jun Hang⁹

Affiliations

¹ Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland, USA.
² US Army Medical Research Institute of Infectious Disease Center for Genome Sciences, Fort Detrick, Maryland, USA.
³ MU Center for Influenza and Emerging Infectious Diseases (CIEID), University of Missouri, Columbia, Missouri, USA.
⁴ Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, Missouri, USA.
⁵ Bond Life Sciences Center, University of Missouri, Columbia, Missouri, USA.
⁶ MU Institute for Data Science and Informatics, University of Missouri, Columbia, Missouri, USA.
⁷ Department of Pathology, School of Medicine, University of Missouri, Columbia, Missouri, USA.
⁸ Department of Electrical Engineering & Computer Science, College of Engineering, University of Missouri, Columbia, Missouri, USA.
⁹ Viral Diseases Branch, Walter Reed Army Institute of Research, Silver Spring, Maryland, USA Jun.hang.civ@mail.mil.

Abstract

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.

Keywords: COVID-19; SARS-CoV-2; acute respiratory illness; emerging infectious viral disease; genomes; next-generation sequencing; pandemic.

This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

COVID-19 / virology
DNA Primers
Genome, Viral*
High-Throughput Nucleotide Sequencing*
Humans
Microfluidics*
RNA, Viral / genetics
Reverse Transcriptase Polymerase Chain Reaction
SARS-CoV-2 / genetics*
Whole Genome Sequencing*

Substances

DNA Primers
RNA, Viral