Overcoming variant mutation-related impacts on viral sequencing and detection methodologies

Abstract

Prompt and accurate pathogen identification, by diagnostics and sequencing, is an effective tool for tracking and potentially curbing pathogen spread. Targeted detection and amplification of viral genomes depends on annealing complementary oligonucleotides to genomic DNA or cDNA. However, genomic mutations that occur during viral evolution may perturb annealing, which can result in incomplete sequence coverage of the genome and/or false negative diagnostic test results. Herein, we demonstrate how to assess, test, and optimize sequencing and detection methodologies to attenuate the negative impact of mutations on genome targeting efficiency. This evaluation was conducted using in vitro-transcribed (IVT) RNA as well as RNA extracted from clinical SARS-CoV-2 variant samples, including the heavily mutated Omicron variant. Using SARS-CoV-2 as a current example, these results demonstrate how to maintain reliable targeted pathogen sequencing and how to evaluate detection methodologies as new variants emerge.

Keywords: COVID; NGS-next generation sequencing; SARS-CoV-2; bioinformatics; infectious disease surveillance and control; molecular diagnostics; variants; viral genomics.

FIGURE 1

SARS-CoV-2 variants necessitate continual assessment of primers utilized for sequencing as variants overlap with primer sites. (A) Timeline representing the emergence of SARS-CoV-2 variants as determined by continual variant monitoring via sequencing. (B) Analysis of Delta and Omicron variants overlapping with primers from several SARS-CoV-2 primer schemes. Orange vertical lines mark mutation locations along variant strains. Blue and red arrowheads mark primer locations and orientations; red arrowheads are primers that overlap with mutation sites in one or more variant strains. (C) Zoom-in of primer-variant overlap analysis in the S gene to N gene region of the genome.

FIGURE 2

SARS-CoV-2 standard and variant genome coverage improves with multi-reference-based primer schemes. Integrative Genome Viewer visualization of read coverage across the SARS-CoV-2 genome (0–4,000 log scale). Genome coverage tracks for ATCC-1986 standard Wuhan-1 cultured RNA control templates in gray, clinical Delta sample templates in blue, and clinical Omicron sample templates in orange. Red arrowheads point to amplicon dropouts with < 100× coverage. The number of bases with less than 100× coverage are listed in the inset beside each genome coverage track. (A) Coverage of the SARS-CoV-2 genomes with ARTICv3 primers. (B) Coverage of SARS-CoV-2 genomes with ARTIC v4, ARTIC v4.1, VarSkip Short, and VarSkip Short v2 primers. (C) Coverage of SARS-CoV-2 genomes with Midnight-1200 and VarSkip Long primers.

FIGURE 3

The Omicron N gene contains a mutation targeted by the 2019-nCoV_N1 detection probe. The CDC 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel includes two primer-probe sets that target the SARS-CoV-2 N gene, named 2019-nCoV_N1 and 2019-nCoV_N2. (A) Visual depiction of Omicron variant mutation within the N1 probe site generated using the Primer Monitor online tool (Tableau worksheet exported from Primer Monitor tool, primer set name “NEB Luna qPCR/CDC”). The Omicron variant from Gauteng, South Africa and the mutation are annotated with a red box and an arrow, respectively. (B) Schematic representation of the two CDC primer-probe sets. Each set includes one forward primer (F), one reverse primer (R), and one fluorescent probe (P). The SARS-CoV-2 Omicron variant has a C to U mutation at position 28,311, which is within the 2019-nCoV_N1 probe (P1) target sequence. Not drawn to scale.

FIGURE 4

The NEB SARS-CoV-2 multiplex assay efficiently detects N gene IVT RNA carrying the Omicron mutation (A–E) and clinical Omicron RNA (F,G) with the CDC 2019-nCoV_N1 primer-probe set. Amplification efficiency in panels (A–D) was evaluated in triplicate over a 7-log range (10⁷-10 copies/reaction) of synthetic N gene RNA for wild-type (black) vs. the Omicron variant (green). Detection sensitivity (E) was evaluated with 10 copies of RNA per reaction with 27 replicates per condition. All reactions tested with 10 copies of input RNA were detected by qPCR. Both the N1 (green) and N2 (blue) targets were efficiently amplified from clinical Omicron RNA samples using the NEB Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit, which includes the CDC 2019-nCoV_N1 and 2019-nCoV_N2 primer-probe sets (F,G). Cq values for each clinical sample shown (G) are included in Supplementary Table 2.