Background: The SARS-CoV-2 virus has assumed considerable importance during the COVID-19 pandemic. Its mutation rate is high, involving the spike (S) gene and thus there has been a rapid spread of new variants. Herein, we describe a rapid, easy, adaptable, and affordable workflow to uniquely identify all currently known variants through as few analyses. Our method only requires two conventional PCRs of the S gene and two Sanger sequencing reactions, and possibly another PCR/sequencing assay on a N gene portion to identify the B.1.160 lineage. Methods: We selected an S gene 1312 bp portion containing a set of SNPs useful for discriminating all variants. Mathematical, statistical, and bioinformatic analyses demonstrated that our choice allowed us to identify all variants even without looking for all related mutations, as some of them are shared by different variants (e.g., N501Y is found in the Alpha, Beta, and Gamma variants) whereas others, that are more informative, are unique (e.g., A57 distinctive to the Alpha variant). Results: A “weight” could be assigned to each mutation that may be present in the selected portion of the S gene. The method’s robustness was confirmed by analyzing 80 SARS-CoV-2-positive samples. Conclusions: Our workflow identified the variants without the need for whole-genome sequencing and with greater reliability than with commercial kits.

Discriminatory Weight of SNPs in Spike SARS-CoV-2 Variants: A Technically Rapid, Unambiguous, and Bioinformatically Validated Laboratory Approach

Musso N.
Conceptualization
;
Bonacci P. G.
Methodology
;
Bongiorno D.
Conceptualization
;
Stracquadanio S.
Conceptualization
;
Bivona D. A.
Validation
;
Fichera M.
Formal Analysis
;
Stefani S.
Writing – Review & Editing
2022-01-01

Abstract

Background: The SARS-CoV-2 virus has assumed considerable importance during the COVID-19 pandemic. Its mutation rate is high, involving the spike (S) gene and thus there has been a rapid spread of new variants. Herein, we describe a rapid, easy, adaptable, and affordable workflow to uniquely identify all currently known variants through as few analyses. Our method only requires two conventional PCRs of the S gene and two Sanger sequencing reactions, and possibly another PCR/sequencing assay on a N gene portion to identify the B.1.160 lineage. Methods: We selected an S gene 1312 bp portion containing a set of SNPs useful for discriminating all variants. Mathematical, statistical, and bioinformatic analyses demonstrated that our choice allowed us to identify all variants even without looking for all related mutations, as some of them are shared by different variants (e.g., N501Y is found in the Alpha, Beta, and Gamma variants) whereas others, that are more informative, are unique (e.g., A57 distinctive to the Alpha variant). Results: A “weight” could be assigned to each mutation that may be present in the selected portion of the S gene. The method’s robustness was confirmed by analyzing 80 SARS-CoV-2-positive samples. Conclusions: Our workflow identified the variants without the need for whole-genome sequencing and with greater reliability than with commercial kits.
2022
Bioinformatic validation
COVID-19
COVID-19 variants
Sanger sequencing
SARS-CoV-2
COVID-19
Clinical Laboratory Techniques
Computational Biology
Coronavirus Nucleocapsid Proteins
Genotype
Humans
Mutation
Phosphoproteins
Polymerase Chain Reaction
Reproducibility of Results
SARS-CoV-2
Sequence Analysis, DNA
Spike Glycoprotein, Coronavirus
Workflow
Polymorphism, Single Nucleotide
File in questo prodotto:
File Dimensione Formato  
viruses-14-00123.pdf

accesso aperto

Tipologia: Versione Editoriale (PDF)
Dimensione 1.15 MB
Formato Adobe PDF
1.15 MB Adobe PDF Visualizza/Apri

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.11769/528757
Citazioni
  • ???jsp.display-item.citation.pmc??? 0
  • Scopus 0
  • ???jsp.display-item.citation.isi??? 0
social impact