SARS-CoV-2 genotyping and sequencing following a simple and economical RNA extraction and storage protocol

• Type of Material: Article
• Authors: Sarah Hernandez, Emory University; Phuong-Vi Nguyen, Emory University; Taz Azmain, Emory University; Anne Piantadosi, Emory University; Jesse Waggoner, Emory University
• Language: English
• Date: 2023-01-19
• Publisher: PUBLIC LIBRARY SCIENCE
• Publication Version: Final Published Version
• Copyright Statement: © 2023 Hernandez et al
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1371/journal.pone.0280577
• Title of Journal or Parent Work: PLOS ONE
• Volume: 18
• Issue: 1
• Start Page: e0280577
• End Page: e0280577
• Grant/Funding Information: This research was supported by an award from the Doris Duke Charitable Foundation (Clinical Scientist Development Award 2019089, JJW; https://www.ddcf.org/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
• Keywords: Science & Technology - Other Topics; Science & Technology; Multidisciplinary Sciences; SWAB
• Research Categories: Health Sciences, Pathology

Abstract

Since the beginning of the SARS-CoV-2 pandemic, supply chain shortages have caused major disruptions in sourcing the materials needed for laboratory-based molecular assays. With increasing demand for molecular testing, these disruptions have limited testing capacity and hindered efforts to mitigate spread of the virus and new variants. Here we evaluate an economical and reliable protocol for the extraction and short-term ambient temperature storage of SARS-CoV-2 RNA. Additional objectives of the study were to evaluate RNA from this protocol for 1) detection of single nucleotide polymorphisms (SNPs) in the spike gene and 2) whole genome sequencing of SARS-CoV-2. The RNAES protocol was evaluated with residual nasopharyngeal (NP) samples collected from Emory Healthcare and Emory Student Health services. All RNAES extractions were performed in duplicate and once with a commercial extraction robot for comparison. Following extraction, eluates were immediately tested by rRT-PCR. SARS-CoV-2 RNA was successfully detected in 56/60 (93.3%) RNAES replicates, and Ct values corresponded with comparator results. Upon testing in spike SNP assays, three genotypes were identified, and all variant calls were consistent with those previously obtained after commercial extraction. Additionally, the SARS-RNAES protocol yield eluate pure enough for downstream whole genome sequencing, and results were consistent with SARS-CoV-2 whole genome sequencing of eluates matched for Ct value. With reproducible results across a range of virus concentrations, the SARS-RNAES protocol could help increase SARS-CoV-2 diagnostic testing and monitoring for emerging variants in resource-constrained communities.