Development of pyrosequencing-based assay for genotyping of different coronavirus variants

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Abstract

BACKGROUND: The rapid spread of SARS-CoV-2 virus, which caused the COVID-19 pandemic, and the emergence of new co-circulating antigenic variants require the development and update of subtyping kits and protocols. Pyrosequencing-based protocols are promising approach for detection of single nucleotide polymorphisms.

AIM: In this study we designed the assays for genotyping Variants of Concern of the SARS-CoV-2 coronavirus using polymerase chain reaction, followed by determination of the virus variant in the sample by pyrosequencing.

MATERIALS AND METHODS: Pyrosequencing assays were designed based on alignment of SARS-CoV-2 sequences. Testing was performed using RNA of SARS-CoV-2 viruses of different lineages (alpha, beta, gamma, delta, and omicron). Pyrosequencing was performed using the PyroMark Q24 system.

RESULTS: The protocols of sample preparation and pyrosequencing were developed and tested for sequencing of regions encoding substitutions in amino acid positions: L18F, T19R, T20N; A67V, Δ69-70; G142D, Δ143-145; Δ156-157, R158G; Δ242-244; K417N/T; L452R; S477N, T478K, E484A/K/Q; H655Y; N679K, P681H/R. The specificity of the system was also evaluated in reactions with a negative control sample (RNA isolated from human nasal swab).

CONCLUSIONS: In this study, we developed and initially tested protocol for detecting coronavirus variants (alpha, beta, gamma, delta, and omicron) from samples collected from cell culture, based on the PCR technique, followed by genotyping of the variants by pyrosequencing with PyroMark Q24. The developed protocols may be adjusted to the current epidemiological situation by increasing the number of detectable sites.

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About the authors

Anna K. Chistyakova

Institute of Experimental Medicine

Author for correspondence.
Email: anna.k.chistiakova@gmail.com
ORCID iD: 0000-0001-9541-5636
SPIN-code: 8852-4103
Scopus Author ID: 57226491888

Student

Russian Federation, Saint Petersburg

Ekaterina A. Stepanova

Institute of Experimental Medicine

Email: fedorova.iem@gmail.com
ORCID iD: 0000-0002-8670-8645
SPIN-code: 8010-3047

Cand. Sci. (Biol.), Leading Research Associate, Department of Virology named after A.A. Smorodintsev

Russian Federation, Saint Petersburg

Irina N. Isakova-Sivak

Institute of Experimental Medicine

Email: isakova.sivak@iemspb.ru
ORCID iD: 0000-0002-2801-1508
SPIN-code: 3469-3600
Scopus Author ID: 23973026600

Dr. Sci. (Biol.), Head of Laboratory of Immunology and Prevention of Viral Infections, A.A. Smorodintsev Department of Virology

Russian Federation, Saint Petersburg

Larisa G. Rudenko

Institute of Experimental Medicine

Email: vaccine@mail.ru
ORCID iD: 0000-0002-0107-9959
SPIN-code: 4181-1372
Scopus Author ID: 7005033248

MD, Dr. Sci. (Med.), Professor, Head of Department of Virology, A.A. Smorodintsev Department of Virology

Russian Federation, Saint Petersburg

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Supplementary files

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2. Fig. 1. The location of the amplified regions matched to the regions of variability in the sequence of the SARS-CoV-2 coronavirus S gene and to the sequence regions encoding different functional regions of the spike protein. The histogram of variability is based on Nextstrain.org data (accessed 24.06.2022) [2]. The variable amino acid residues characterizing Variants of Concern are designated. The functional parts of S protein are defined according to [16]: NTD (N-terminal domain), RBD — receptor-binding domain, RBM — receptor-binding motif, SD1/2 – subdomain 1, 2, FP — fusion peptide, HR1 — heptad repeat 1, CH – central helix, CD — connection domain, HR2 — heptad repeat 2, TM — transmembrane domain, CT — cytoplasmic tail

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3. Fig. 2. Pyrograms obtained by pyrosequencing the genetic material of different SARS-CoV-2 variants according to the protocol with the F28 sequencing primer. On the left is the subtype of the virus in the sample, on the right is the sequence of the site (decoding of the pyrogram). OKO is a negative control sample, human epithelial cell scraping material that does not contain coronavirus genetic material

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4. Fig. 1. Pyrograms obtained by pyrosequencing the genetic material of different variants of coronaviruses according to the protocol with the F179 sequencing primer: a — reference strain; b — alpha variant (Δ69-70); c — omicron variant (A67V, Δ69-70); d — scraping material of human epithelial cells. In each fragment of the figure, the top shows the shape of the distribution of peaks calculated on the basis of the genetic sequence, and the bottom shows the resulting pyrograms

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5. Fig. 2. Pyrograms obtained by pyrosequencing the genetic material of different variants of coronaviruses according to the protocol with the F406 sequencing primer: a — reference strain, b — alpha variant (Δ144); c — delta variant (G142D); d — omicron variant (G142D, Δ143-145); e — scraping material of human epithelial cells. On each fragment of the figure, the shape of the distribution of peaks calculated on the basis of the genetic sequence is shown on top, and the resulting pyrograms are shown below

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6. Fig. 3. Pyrograms obtained by pyrosequencing the genetic material of different variants of coronaviruses according to the protocol with the F447 sequencing primer: a — reference strain; b — beta variant (Δ156-157, R158G); c — omicron variant; d — scraping material of human epithelial cells. On each fragment of the figure, the shape of the distribution of peaks calculated on the basis of the genetic sequence is shown on top, and the resulting pyrograms are shown below

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7. Fig. 4. Pyrograms obtained by pyrosequencing the genetic material of different variants of coronaviruses according to the protocol with the R750 sequencing primer: a — reference strain; b — beta variant (Δ242-244); c — omicron variant; d — scraping material of human epithelial cells. On each fragment of the figure, the shape of the distribution of peaks calculated on the basis of the genetic sequence is shown on top, and the resulting pyrograms are shown below

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8. Fig. 5. Pyrograms obtained by pyrosequencing of the genetic material of different variants of coronaviruses according to the protocol with the sequencing primer F1231: a — reference strain; b — beta variant (K417N); c — gamma variant (K417T); d — omicron variant (K417N); e — scraping material of human epithelial cells. On each fragment of the figure, the shape of the distribution of peaks calculated on the basis of the genetic sequence is shown at the top, and the obtained pyrograms are shown at the bottom. Non-specific primer annealing and non-specific peaks on the pyrogram are observed, optimization of the study is required

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9. Fig. 6. Pyrograms obtained by pyrosequencing the genetic material of different variants of coronaviruses according to the protocol with the sequencing primer F1328: a — reference strain; b — delta variant (L452R); c — omicron variant; d — scraping material of human epithelial cells. In each fragment of the figure, the top shows the shape of the distribution of peaks calculated on the basis of the genetic sequence, and the bottom shows the resulting pyrograms

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10. Fig. 7. Pyrograms obtained by pyrosequencing the genetic material of different variants of coronaviruses according to the protocol with the sequencing primer F1410: a — reference strain; b — delta variant (T478K); c — omicron variant (S477N, T478K); d — scraping material of human epithelial cells. In each fragment of the figure, the top shows the shape of the distribution of peaks calculated on the basis of the genetic sequence, and the bottom shows the resulting pyrograms

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11. Fig. 8. Pyrograms obtained by pyrosequencing the genetic material of different variants of coronaviruses according to the protocol with the F1431 sequencing primer: a — reference strain; b — gamma variant (E484K); c — delta variant; d — omicron variant (E484A); e — human epithelial cell scraping material. In each fragment of the figure, the top shows the shape of the distribution of peaks calculated on the basis of the genetic sequence, and the bottom shows the resulting pyrograms

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12. Fig. 9. Pyrograms obtained by pyrosequencing the genetic material of different variants of coronaviruses according to the protocol with the R1986 sequencing primer: a — reference strain; b — gamma variant (H655Y); c — omicron variant (H655Y); d — scraping material of human epithelial cells. In each fragment of the figure, the top shows the shape of the distribution of peaks calculated on the basis of the genetic sequence, and the bottom shows the resulting pyrograms

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13. Fig. 10. Pyrograms obtained by pyrosequencing of the genetic material of different variants of coronaviruses according to the protocol with the sequencing primer R2064: a — reference strain; b — alpha variant (P681H); c — delta variant (P681R); d — scraping material of human epithelial cells. In each fragment of the figure, the top shows the shape of the distribution of peaks calculated on the basis of the genetic sequence, and the bottom shows the resulting pyrograms

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