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Optimization of the SARS-CoV-2 ARTIC network V4 primers and whole genome sequencing protocol

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Lambisia, Arnold W., Mohammed, Khadija S., Makori, Timothy O., Ndwiga, Leonard, Mburu, Maureen W., Morobe, John M. , Moraa, Edidah O., Musyoki, Jennifer, Murunga, Nickson, Mwangi, Jane N., Nokes, D. James, Agoti, Charles N., Ochola-Oyier, Lynette Isabella and Githinji, George (2022) Optimization of the SARS-CoV-2 ARTIC network V4 primers and whole genome sequencing protocol. Frontiers in Medicine, 9 . 836728. doi:10.3389/fmed.2022.836728 ISSN 2296-858X.

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Official URL: https://doi.org/10.3389/fmed.2022.836728

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Abstract

Introduction: The ARTIC Network's primer set and amplicon-based protocol is one of the most widely used SARS-CoV-2 sequencing protocol. An update to the V3 primer set was released on 18th June 2021 to address amplicon drop-off observed among the Delta variant of concern. Here, we report on an in-house optimization of a modified version of the ARTIC Network V4 protocol that improves SARS-CoV-2 genome recovery in instances where the original V4 pooling strategy was characterized by amplicon drop-offs.

Methods: We utilized a matched set of 43 clinical samples and serially diluted positive controls that were amplified by ARTIC V3, V4 and optimized V4 primers and sequenced using GridION from the Oxford Nanopore Technologies'.

Results: We observed a 0.5% to 46% increase in genome recovery in 67% of the samples when using the original V4 pooling strategy compared to the V3 primers. Amplicon drop-offs at primer positions 23 and 90 were observed for all variants and positive controls. When using the optimized protocol, we observed a 60% improvement in genome recovery across all samples and an increase in the average depth in amplicon 23 and 90. Consequently, ≥95% of the genome was recovered in 72% (n = 31) of the samples. However, only 60–70% of the genomes could be recovered in samples that had <28% genome coverage with the ARTIC V3 primers. There was no statistically significant (p > 0.05) correlation between Ct value and genome recovery.

Conclusion: Utilizing the ARTIC V4 primers, while increasing the primer concentrations for amplicons with drop-offs or low average read-depth, greatly improves genome recovery of Alpha, Beta, Delta, Eta and non-VOC/non-VOI SARS-CoV-2 variants.

Item Type: Journal Article
Subjects: Q Science > QH Natural history
R Medicine > RA Public aspects of medicine
Divisions: Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- )
SWORD Depositor: Library Publications Router
Library of Congress Subject Headings (LCSH): COVID-19 (Disease), COVID-19 (Disease) -- Epidemiology -- Data processing , Human gene mapping , Genomes -- Analysis
Journal or Publication Title: Frontiers in Medicine
Publisher: Frontiers Media S.A.
ISSN: 2296-858X
Official Date: 17 February 2022
Dates:
DateEvent
17 February 2022Published
26 January 2022Accepted
Volume: 9
Article Number: 836728
DOI: 10.3389/fmed.2022.836728
Status: Peer Reviewed
Publication Status: Published
Access rights to Published version: Open Access (Creative Commons)
Date of first compliant deposit: 16 March 2022
Date of first compliant Open Access: 18 March 2022
RIOXX Funder/Project Grant:
Project/Grant IDRIOXX Funder NameFunder ID
17/63/82National Institute for Health Researchhttp://dx.doi.org/10.13039/501100000272
16/136/33 National Institute for Health Researchhttp://dx.doi.org/10.13039/501100000272
102975Foreign, Commonwealth and Development OfficeUNSPECIFIED
220985Foreign, Commonwealth and Development OfficeUNSPECIFIED
102975Wellcome Trusthttp://dx.doi.org/10.13039/100010269
220985Wellcome Trusthttp://dx.doi.org/10.13039/100010269
UNSPECIFIEDAfrica CDChttp://viaf.org/viaf/365161575628504730008
UNSPECIFIEDAfrican Society for Laboratory Medicinehttp://viaf.org/viaf/15151170923739090128
UNSPECIFIEDWorld Health Organization. Regional Office for Africahttp://viaf.org/viaf/135480005
17/63/82 (GeMVi)National Institute for Health Researchhttp://dx.doi.org/10.13039/501100000272
16/136/33 (TIBA)National Institute for Health Researchhttp://dx.doi.org/10.13039/501100000272
DEL-15- 003Africa Initiative (Program)http://viaf.org/viaf/170846325
107769/Z/10/ZWellcome Trusthttp://dx.doi.org/10.13039/100010269
UNSPECIFIEDGreat Britain. Governmenthttp://viaf.org/viaf/124906873

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