SNP and Phylogenetic Characterization of Low Viral Load SARS-CoV-2 Specimens by Target Enrichment

Gregory S Orf,Illya Mowerman,Todd V Meyer,Matthew L Faron,Cheryl Jennings,Kenn Forberg,Aurash Mohaimani,Michael G Berg,Alan L Landay,Amy S Fox,D Yitz Goldstein,Gavin A Cloherty

doi:10.3389/fviro.2021.765974

Abstract

Background: Surveillance of SARS-CoV-2 across the globe has enabled detection of new variants and informed the public health response. With highly sensitive methods like qPCR widely adopted for diagnosis, the ability to sequence and characterize specimens with low titers needs to keep pace.Methods: Nucleic acids extracted from nasopharyngeal swabs collected from four sites in the United States in early 2020 were converted to NGS libraries to sequence SARS-CoV-2 genomes using metagenomic and xGen target enrichment approaches. Single nucleotide polymorphism (SNP) analysis and phylogeny were used to determine clade assignments and geographic origins of strains.Results: SARS-CoV-2-specific xGen enrichment enabled full genome coverage for 87 specimens with Ct values &lt;29, corresponding to viral loads of &gt;10,000 cp/ml. For samples with viral loads between 103 and 106 cp/ml, the median genome coverage for xGen was 99.1%, sequence depth was 605X, and the “on-target” rate was 57 ± 21%, compared to 13%, 2X and 0.001 ± 0.016%, respectively, for metagenomic sequencing alone. Phylogenetic analysis revealed the presence of most clades that existed at the time of the study, though clade GH dominated in the Midwest.Conclusions: Even as vaccines are being widely distributed, a high case load of SARS-CoV-2 infection persists around the world. Viral genetic surveillance has succeeded in warning the public of new variants in circulation and ensured that diagnostic tools remain resilient to a steadily increasing number of mutations. Target capture offers a means of characterizing low viral load samples which would normally pose a challenge for metagenomic sequencing.

Highlights

Since the first cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were reported in late 2019 in China, nearly 205 million cases of coronavirus disease 2019 (COVID-19) and 4.3 million COVID-19 deaths have been reported
One-hundred ninety-four nasopharyngeal swabs in viral transfer medium (VTM) with positive SARS-CoV-2 diagnostic tests were collected from several clinical sites in the United States between March 2020 and June 2020
The remaining 8% tested negative in our laboratory by the Abbott RealTime SARS-CoV-2 qPCR assay, even though they tested positive at the originating laboratory

Summary

Introduction

Since the first cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were reported in late 2019 in China, nearly 205 million cases of coronavirus disease 2019 (COVID-19) and 4.3 million COVID-19 deaths have been reported (as of August 2021; Johns Hopkins University, https:// coronavirus.jhu.edu/). In late 2020 and early 2021, new SARS-CoV-2 variants linked to heightened transmissibility have been reported almost monthly, such as Alpha, Beta, Gamma, and Delta, revealing the need for constant and rapid surveillance to characterize mutations and evaluate their effects on infection patterns and vaccine efficacy [9,10,11,12]. A tremendous amount of sequencing information has been generated for SARS-CoV-2, with well over 1 million sequences deposited in GISAID and the NCBI as of August 2021 (https:// www.gisaid.org/ and https://www.ncbi.nlm.nih.gov/sars-cov-2/). These sequences are generally >99.9% identical, distinguished by only a few SNPs [13]. With highly sensitive methods like qPCR widely adopted for diagnosis, the ability to sequence and characterize specimens with low titers needs to keep pace

Methods

Results

Discussion

Conclusion