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

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 <29, corresponding to viral loads of >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.

SARS-CoV-2 RNA Is Readily Detectable at Least 8 Months after Shedding in an Isolation Facility

“…Since many factors can cause variation in sequencing runs, depth of coverage was normalized within each sample. As seen with fresh SARS-CoV-2 samples, there was variation in depth of coverage across the genome ( 10 , 11 ). Overall, the depths of coverage for the samples paralleled each other and demonstrated that complete SARS-CoV-2 genomes could be assembled from surface samples collected at least 74 days after the room had been occupied.…”

Next-generation sequencing survey of acute febrile illness in Senegal (2020–2022)

“…Total nucleic acids (TNA) were extracted from 500 μl of each Benzonase-treated plasma specimen on an m 2000sp system using a laboratory-defined assay (Abbott Molecular, Des Plaines, IL). The automated extraction protocol uses magnetic microparticles for capture and modified m 2000 buffers to recover both RNA and DNA ( Orf et al, 2021 ). Each extraction contained four positive controls (viral stocks spiked into HIV-positive plasma at 3.0 log copies/ml), one negative control (normal human plasma), and one no-template control (phosphate-buffered saline) per ninety primary specimens.…”

“…A major benefit of this technology is its ability to multiplex large cohorts of specimens ( Radford et al, 2012 ; Chiu, 2013 ; Kapuscinski et al, 2021 ; Ko et al, 2022 ). This can be performed in an unbiased fashion (metagenomics; mNGS) to surveille or discover any pathogen present in a specimen ( Chiu and Miller, 2019 ; Ko et al, 2022 ), or it can be targeted via targeted enrichment (teNGS) to expedite identification of known pathogens, even at low concentration ( O’Flaherty et al, 2018 ; Yamaguchi et al, 2018 ; Deng et al, 2020 ; Orf et al, 2021 ).…”

The Principles of SARS-CoV-2 Intervariant Competition Are Exemplified in the Pre-Omicron Era of the Colombian Epidemic

“…Each data set was analyzed using a local, sandboxed Docker deployment of Nextclade ( 55 , 57 ). Genomes with “bad” quality control (QC) scores (using the default scoring metrics in Nextclade) or >50 total mutations were discarded.…”