Robust and highly multiplexed assay for identification of SARS-CoV-2 variants

In a recent study posted to the medRxiv* preprint server, researchers compared the performance of a highly multiplexed mass spectrometry-based commercial assay to identify severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants.

Study: A robust, highly multiplexed mass spectrometry assay to identify SARS-CoV-2 variants. Image Credit: Design_Cells/Shutterstock
Study: A robust, highly multiplexed mass spectrometry assay to identify SARS-CoV-2 variants. Image Credit: Design_Cells/Shutterstock


The genomically diverse SARS-CoV-2 variants differ in their transmissibility, pathogenicity, and responses to anti-SARS-CoV-2 therapeutic agents. Therefore, the variants pose a unique threat to diagnostic laboratories and healthcare systems and must be identified for improved coronavirus disease 2019 (COVID-19) surveillance, SARS-CoV-2 prevention, and standard of care for COVID-19 patients.

For variant identification, whole-genome sequencing (WGS) is the gold standard but is not widely accessible to clinical and microbiological laboratories as it is technique sensitive, requires infrastructure, and is expensive. This warrants the need to develop other robust, high-resolution, and highly multiplexed SARS-CoV-2 assays such as molecular assays to accurately detect SARS-CoV-2 infected individuals.

About the study

In the present study, researchers highlighted the diagnostic capabilities of the highly multiplexed and commercial Agena MassARRAY® SARS-CoV-2 Variant Panel v3 assay in identifying SARS-CoV-2 variants which circulated between September 2, 2020, and March 2, 2022.

For the analysis, 391 SARS-CoV-2 clinical ribonucleic acid (RNA) samples were obtained across health systems in Bogotá, Colombia, and New York City (NYC). The samples had been previously subjected to WGS analysis, the data of which was used for comparative evaluation of the diagnostic performance of the Agena MassARRAY® SARS-CoV-2 variant panel v3. The samples comprised 349 upper respiratory tract [e.g., nasopharyngeal (n=43) anterior nares] and saliva specimens.

SARS-CoV-2 RNA was extracted from the samples and subsequently subjected to reverse transcription-polymerase chain reaction (RT-PCR) analysis and next-generation sequencing, followed by genomic assembling and PANGO (phylogenetic assignment of named global outbreak lineages) assignments. Sequence libraries were prepared by performing long-read Oxford Nanopore MinION sequencing.

The panel comprised integrated RT-PCR and MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) for detecting targeted polymorphisms in the SARS-CoV-2 spike (S) gene. The diverse set of SARS-CoV-2 variants was examined for the level of interrater agreement and diagnostic specificity and sensitivity across the variants on the molecular panel and across the S gene targets. Furthermore, SARS-CoV-2 genomes on the Global Initiative on Sharing Avian Influenza Data (GISAID) database (which was last accessed on 6 May 2022) were assessed to determine the prevalence of variant panel targets across the Omicron subvariants.


Out of 391 specimens, 381 single variant genomic sequences were identified, whereas the ten remaining specimens yielded mixed genomic assemblies and, thus, an inconclusive result. RNA extracted from the samples corresponded to 12 out of 16 variant calls on the molecular panel which included Iota (n=39), Alpha (n=40), Delta (n = 3), Delta AY.x subvariant (n = 107), and Omicron BA.1 (n=79). However, only 11 out of 12 variant calls (excluding the D614G strain) were used for the agreement analysis.

Only 62 (out of 391 specimens) were observed for which the variant calls were in disagreement with WGS analysis, of which the sequenced variant of 45 specimens did not have a defined variant algorithm on the molecular panel. As a result, only 17 (5%) of the RNA samples yielded results that differed from those of WGS analysis. Near-perfect interrater agreement levels were observed between WGS and the assay for 9 out of 11 variant calls and 25 out of 30 targets tested on the molecular panel. The Eta and Zeta variant calls showed the least interrater agreement. Polymorphic targets with suboptimal agreement levels included D215G, L242_244del, N501Y, and K1191N.

The assay demonstrated high diagnostic sensitivity (≥93.7%) for contemporary variants (e.g., Iota, Alpha, Delta variants, and Omicron BA.1 subvariant) and high diagnostic specificity for all the 11 variants tested (≥96%) and all 30 targets (≥94%) tested. The targets with the lowest sensitivities included D80A, D215G, L242_244del, and N501Y. Further, after excluding the Zeta variant, the panel demonstrated high positive predictive values (≥0.9) and high negative predictive values (≥0.984) for all the variant calls.

Of interest, for the N501Y target, 79 out of 158 samples with WGS-identified polymorphisms yielded false-negative results on the variant panel, all of which belonged to the Omicron BA.1 subvariant, and on excluding the BA.1 samples, near-perfect (kappa = 0.98) interrater agreement was observed. This indicated that genomic variations beyond the scope of the assay’s design could provide distinctive targets for new variants.

All nine Omicron BA.2 specimens generated a distinct target signature on the panel assay comprising N501Y, S477N, T478K, P681H, and D614G targets and N439K target dropout. All BA.2 specimens harbored the T22882G polymorphism. Almost all BA.2.12.1 genomes harbored the S477N, K417N, T478K, P681H, D614G, and N501Y substitutions and N439K target dropout. Over 70% of BA.3 genomes encoded the S477N, T478, N501Y, D614G, and P681H substitutions. About 88% and 99% of BA.4 and 9 BA.5 genomes encoded the L452R substitution, respectively.


Overall, the study findings exemplified the power of highly multiplexed diagnostic SARS-CoV-2 variant panels for accurate variant calling and highlighted distinct target patterns that can be utilized to identify variants not yet defined on the panel, including Omicron BA.2.

*Important notice

medRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
Pooja Toshniwal Paharia

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Pooja Toshniwal Paharia

Dr. based clinical-radiological diagnosis and management of oral lesions and conditions and associated maxillofacial disorders.


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