Analysis of the ARTIC V4 and V4.1 SARS-CoV-2 primers and their impact on the detection of Omicron BA.1 and BA.2 lineage-defining mutations

Fatima R. Ulhuq; Madhuri Barge; Kerry Falconer; Jonathan Wild; Goncalo Fernandes; Abbie Gallagher; Suzie McGinley; Ahmad Sugadol; Muhammad Tariq; Daniel Maloney; Juliet Kenicer; Rebecca Dewar; Kate Templeton; Martin P. McHugh

doi:10.1099/mgen.0.000991

Volume 9, Issue 4

Research Article

Open Access

Analysis of the ARTIC V4 and V4.1 SARS-CoV-2 primers and their impact on the detection of Omicron BA.1 and BA.2 lineage-defining mutations

Fatima R. Ulhuq¹, Madhuri Barge¹, Kerry Falconer¹, Jonathan Wild¹, Goncalo Fernandes¹, Abbie Gallagher¹, Suzie McGinley¹, Ahmad Sugadol¹, Muhammad Tariq¹, Daniel Maloney^1,2, Juliet Kenicer¹, Rebecca Dewar¹, Kate Templeton¹ and Martin P. McHugh^1,3
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Affiliations: ¹ Viral Genotyping Reference Laboratory, Royal Infirmary of Edinburgh, NHS Lothian, Edinburgh, EH16 4SA, UK ² Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, EH9 3JR, UK ³ School of Medicine, University of St Andrews, St Andrews, KY16 9TF, UK
*Correspondence: Fatima R. Ulhuq, [email protected] *Correspondence: Martin P. McHugh, [email protected]
Published: 21 April 2023 https://doi.org/10.1099/mgen.0.000991

Abstract

The ARTIC protocol uses a multiplexed PCR approach with two primer pools tiling the entire SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) genome. Primer pool updates are necessary for accurate amplicon sequencing of evolving SARS-CoV-2 variants with novel mutations. The suitability of the ARTIC V4 and updated V4.1 primer scheme was assessed using whole genome sequencing of Omicron from clinical samples using Oxford Nanopore Technology. Analysis of Omicron BA.1 genomes revealed that 93.22 % of clinical samples generated improved genome coverage at 50× read depth with V4.1 primers when compared to V4 primers. Additionally, the V4.1 primers improved coverage of BA.1 across amplicons 76 and 88, which resulted in the detection of the variant-defining mutations G22898A, A26530G and C26577G. The Omicron BA.2 sub-variant (VUI-22JAN-01) replaced BA.1 as the dominant variant by March 2022, and analysis of 168 clinical samples showed reduced coverage across amplicons 15 and 75. Upon further interrogation of primer binding sites, a mutation at C4321T [present in 163/168 (97 %) of samples] was identified as a possible cause of complete dropout of amplicon 15. Furthermore, two mutations were identified within the primer binding regions for amplicon 75: A22786C (present in 90 % of samples) and C22792T (present in 12.5 % of samples). Together, these mutations may result in reduced coverage of amplicon 75, and further primer updates would allow the identification of the two BA.2-defining mutations present in amplicon 75: A22688G and T22679C. This work highlights the need for ongoing surveillance of primer matches as circulating variants evolve and change.

Received: 05/12/2022
Accepted: 22/02/2023
Published Online: 21/04/2023

Keyword(s): BA.1 , BA.2 , Nanopore , Omicron , SARS-CoV-2 and whole genome sequencing

This is an open-access article distributed under the terms of the Creative Commons Attribution License.

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Analysis of the ARTIC V4 and V4.1 SARS-CoV-2 primers and their impact on the detection of Omicron BA.1 and BA.2 lineage-defining mutations

M Gen 9, 000991 (2023); https://doi.org/10.1099/mgen.0.000991

/content/journal/mgen/10.1099/mgen.0.000991

Volume 9, Issue 4

Research Article

Open Access

Analysis of the ARTIC V4 and V4.1 SARS-CoV-2 primers and their impact on the detection of Omicron BA.1 and BA.2 lineage-defining mutations

Abstract

Supplementary material 1

Supplementary material 2

Supplementary material 3

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