Combined Use of RT-qPCR and NGS for Identification and Surveillance of SARS-CoV-2 Variants of Concern in Residual Clinical Laboratory Samples in Miami-Dade County, Florida

Abstract

Over the course of the COVID-19 pandemic, SARS-CoV-2 variants of concern (VOCs) with increased transmissibility and immune escape capabilities, such as Delta and Omicron, have triggered waves of new COVID-19 infections worldwide, and Omicron subvariants continue to represent a global health concern. Tracking the prevalence and dynamics of VOCs has clinical and epidemiological significance and is essential for modeling the progression and evolution of the COVID-19 pandemic. Next generation sequencing (NGS) is recognized as the gold standard for genomic characterization of SARS-CoV-2 variants, but it is labor and cost intensive and not amenable to rapid lineage identification. Here we describe a two-pronged approach for rapid, cost-effective surveillance of SARS-CoV-2 VOCs by combining reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) and periodic NGS with the ARTIC sequencing method. Variant surveillance by RT-qPCR included the commercially available TaqPath COVID-19 Combo Kit to track S-gene target failure (SGTF) associated with the spike protein deletion H69-V70, as well as two internally designed and validated RT-qPCR assays targeting two N-terminal-domain (NTD) spike gene deletions, NTD156-7 and NTD25-7. The NTD156-7 RT-qPCR assay facilitated tracking of the Delta variant, while the NTD25-7 RT-qPCR assay was used for tracking Omicron variants, including the BA.2, BA.4, and BA.5 lineages. In silico validation of the NTD156-7 and NTD25-7 primers and probes compared with publicly available SARS-CoV-2 genome databases showed low variability in regions corresponding to oligonucleotide binding sites. Similarly, in vitro validation with NGS-confirmed samples showed excellent correlation. RT-qPCR assays allow for near-real-time monitoring of circulating and emerging variants allowing for ongoing surveillance of variant dynamics in a local population. By performing periodic sequencing of variant surveillance by RT-qPCR methods, we were able to provide ongoing validation of the results obtained by RT-qPCR screening. Rapid SARS-CoV-2 variant identification and surveillance by this combined approach served to inform clinical decisions in a timely manner and permitted better utilization of sequencing resources.

Keywords: COVID-19; RT-qPCR; SARS-CoV-2; VOC; delta; surveillance.

Figure 1

Impact of Ct Value on ARTIC SARS-CoV-2 Sequencing Performance. A retrospective analysis showing 126/246 of sequenced samples with N-gene target Ct values ranging from 30 to 35 (TaqPath COVID-19 Combo Kit RT-qPCR assay) failed, representing a sequencing failure rate of 51%. This failure rate was significantly decreased in samples with Ct values < 30, where only 54/1404 (4%) resulted in sequencing failure.

Figure 2

Correlation Between ARTIC SARS-CoV-2 Sequencing Failure Rate and Sample Ct. To determine the impact of Ct value on NGS performance with the ARTIC SARS-CoV-2 sequencing method, 246 sample that failed NGS were grouped into Ct value ranges of 30.1–30.9, 31.0–31.9, 32.0–32.9, 33.0–33.9, and 34.0–34.9. Sequencing failure rate increased in a Ct-dependent manner (correlation R² = 0.999), ranging from 28% (15/54 samples, Ct 30.1–30.9) to 83% (25/30 samples, Ct 34–34.9).

Figure 3

RT-qPCR target deletions for VOCs Delta and Omicron. (a) Alignment of Delta E156-F157 deletion with Wuhan reference sequence. This 6 bp deletion (Delta (Mut.)) spans three codons and results in the deletion of amino acids glutamate (E) at position 156 and phenylalanine (F) at position 157, and a non-synonymous substitution at position 158 (arginine (R) for glycine (G)). (b) Alignment of Omicron BA.2 lineage P25-P26-A27 deletion (also present in BA.4 and BA.5 lineages) with Wuhan reference sequence. The 9 bp deletion spans four codons resulting in a non-synonymous substitution of leucine (L) with serine (S) at position 24, and deletion of two proline (P) and one alanine (A) residues at positions 25–27. Ref. aa (reference amino acids), Ref. seq (reference sequence), Mut. (Mutation), Sub aa (substitution amino acids), Nuc. (nucleotides).

Figure 4

SARS-CoV-2 VOC Surveillance Workflow. In the workflow depicted, nasopharyngeal swab (NPS) samples were obtained from Jackson Health System (JHS), University of Miami Health System (UHealth), and University of Miami (UM) for variant identification. The first RT-qPCR assay (TaqPath) serves to identify SGTF/SGTL associated with the H69-V70 deletion present in the Omicron BA.1, BA.4, and BA.5 lineages. For this assay, our N gene target Ct cutoff was 35, with similar amplification (<2 Ct difference) for the ORF1ab and S gene targets in samples showing S gene target amplification (SGTA) and the same rule applied for the ORF1ab gene target in SGTF/SGTL samples. Next, samples (SGTF/SGTL and SGTA) were screened with the NTD25-7 RT-qPCR assay, for this and the Delta assay we were able to increase the Ct cutoff to 38 as we found reliable and reproducible results within this Ct range. Samples positive with the NTD25-7 assay, which also exhibit SGTF/SGTL, were presumed BA.4 or BA.5 lineages and were subsequently sequenced for lineage confirmation. SGTF/SGTL samples negative with the NTD25-7 assay are presumed BA.1 lineage. SGTA samples positive with the NTD25-7 assay were presumed BA.2 lineage. BA.2-negative, SGTA samples were subsequently screened for the Delta variant by targeted RT-qPCR (NTD156-7). If negative for Delta, these samples were sequenced for lineage identification.

Figure 5

SARS-COV-2 Variant Proportions by RT-qPCR and NGS. Variant proportions were determined by RT-qPCR and NGS in residual COVID-19-positive samples collected from week 1 (1/3–1/9) to week 13 (3/23–4/1), 2022. Variant surveillance by RT-qPCR (n = 976) was performed weekly using the TaqPath assay in combination with internally designed and validated targeted RT-qPCR assays (NTD156-7 and NTD25-7). Subsets of these samples (n = 169) (Ct < 30) were sequenced on weeks 3 (n = 39), 5 (n = 14), 8 (n = 40), 10 (n = 48), and 12 (n = 28). Periodic confirmation by NGS of results obtained by RT-qPCR screenings provided confidence in the ongoing variant surveillance by targeted RT-qPCR methods. For the five sequencing timepoints illustrated, concordance between NGS and RT-qPCR results was 100%. SGTA (S-gene target amplification detected with the TaqPath assay); NTD25-7 (RT-qPCR assay targeting the N-terminal-domain deletion corresponding to amino acids 25–27 of the spike (S) protein in Omicron BA.2 lineages); SGTF/SGTL (S-gene target failure/S-gene target late amplification (TaqPath) corresponding to NTD deletion 69–70 of the S protein in Omicron BA.1 lineages); NTD156-7 (RT-qPCR assay targeting the NTD deletion corresponding to amino acids 156–157 of the S protein in the Delta lineage).