Sequencing SARS-CoV-2 from antigen tests

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). In early 2021, many clinicians transitioned to antigen-based SARS-CoV-2 detection tests, which use anterior nasal swabs for SARS-CoV-2 antigen detection. Despite this shift in testing methods, the need for whole-genome sequence surveillance remains. Thus, we developed a workflow for whole-genome sequencing with antigen test-derived swabs as an input rather than nasopharyngeal swabs. In this study, we use excess clinical specimens processed using the BinaxNOW™ COVID-19 Ag Card. We demonstrate that whole-genome sequencing from antigen tests is feasible and yields similar results from RT-PCR-based assays utilizing a swab in viral transport media.

Fig 1. Assay development.

(A) An NP specimen was used to evaluate the feasibility of obtaining viral RNA from Ag-card derived specimens. RT-PCR was carried out on a positive control and on mock Ag-card specimens collected under each condition. (B) RT-PCR N1 and RP Ct values, sequence library quality scores, and viral PANGO lineage assignments of antigen card specimens and reference NP specimen #5195. (C) IGV screen shots of sample SARS-CoV-2 genome coverage. VR-1986D = Positive Control Genomic RNA from SARS-Cov-2, Isolate USA-WA1/2020.

Fig 2. Sample source performance comparison.

(A) SARS-CoV-2 positive BinaxNOW™ COVID-19 Ag Cards were used to evaluate which part of the card, swab or lateral flow positive line, yielded the highest quantity and quality of viral RNA. Extracted RNA was tested for N1 and RP levels using RT-PCR. The average N1 and RP C_T values are plotted. n = 7 cards. Statistical analysis = Two-tailed, paired t-test, * p<0.05; *** p<0.0005 (B) N1 and RP Ct values, library quality scores and PANGO lineage assignments. (C) IGV screen shots of SARS-CoV-2 genome coverage.