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Sequencing SARS-CoV-2 from Antigen Tests

Ashley Nazario-Toole, Holly M. Nguyen, Hui Xia, Dianne N. Frankel, John W. Kieffer, Thomas F. Gibbons

Abstract
Genomic surveillance empowers agile responses to SARS-CoV-2 by enabling scientists and public health analysts to issue recommendations aimed at slowing transmission, prioritizing contact tracing, and building a robust genomic sequencing surveillance strategy. Since the start of the pandemic, real time RT-PCR diagnostic testing from upper respiratory specimens, such as nasopharyngeal (NP) swabs, has been the standard. Moreover, respiratory samples in viral transport media are the ideal specimen for SARS-CoV-2 whole-genome sequencing (WGS).

Introduction
As of January 2022, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has resulted in over 5.5 million deaths worldwide, with more than 836,000 deaths in the United States of America alone. Due to the continuing evolution of the pandemic, ongoing genomic surveillance of SARS-CoV-2 is critical for identifying emerging variants, confirming cases of reinfection, and vaccine breakthrough cases, all of which provide information to be considered when implementing any changes to public health policies.

Materials and Methods:

Specimen acquisition
Specimens were collected under an exempt research protocol approved by the 59th MDW Institutional Review Board (IRB # FHW20200103E) with a waiver of informed consent for the use of residual specimens for sequencing and epidemiological studies. The BinaxNOW™ COVID-19 Ag Card (Abbott) was used to test basic medical trainees for SARS-CoV-2.

Specimen preparation
The antigen card swabs were gently removed from completed tests, placed in a 15 mL conical tube with 500 μL DNA/RNA Shield (Zymo Research, Catalog #R1100), and stored at 4°C until RNA extraction.

RNA extraction
Samples were extracted using the EZ1 Virus Mini Kit v2.0 (Qiagen, Catalog # 955134), per the manual. 400 μL of sample was extracted and eluted into 60 μL AVE buffer (supplied in the kit). The following controls were run with each extraction: (1) positive control, which was a nasopharyngeal swab of SARS-CoV-2 diluted to achieve a CT of approximately 25, and (2) a negative control, which was 200 μL nuclease-free water and 200 μL of DNA/RNA Shield. The extracted RNA was frozen at -80°C or used immediately for RT-PCR.

Discussion
Unlike previous pandemics, global genomic surveillance of SARS-CoV-2 has occurred in near real-time. Medical, DoD, and government institutions actively rely on genomic sequencing data to support public health decisions, underscoring the need for continued sequencing as the virus and pandemic evolve.

Acknowledgments: The authors would like to thank the laboratories who provided the excess clinical specimens used in this research.

Citation: Nazario-Toole A, Nguyen HM, Xia H, Frankel DN, Kieffer JW, Gibbons TF (2022) Sequencing SARS-CoV-2 from antigen tests. PLoS ONE 17(2): e0263794. https://doi.org/10.1371/journal.pone.0263794

Editor: RuslanKalendar, University of Helsinki: HelsinginYliopisto, FINLAND

Received: August 19, 2021; Accepted: January 26, 2022; Published: February 8, 2022.

Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Data Availability: All data and metadata underlying the reported findings have been deposited in the NCBI SRA and GISAID EpiCoV public data repositories. Miseq FASTQ files are published under SRA BioProject accession # PRJNA781677. FASTA files for each SARS-CoV-2 isolate are published on the GISAID EpiCoV Database under accession numbers EPI_ISL_6473528; EPI_ISL_6474667; EPI_ISL_6475107; EPI_ISL_6475110.

Funding: This study was funded 59th Medical Wing Clinical Investigations Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The views expressed are those of the authors and do not reflect the official views of the Department of Defense or its Components.

Competing interests: The authors have declared that no competing interests exist.

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