Optimization of the SARS-CoV-2 ARTIC Network V4 Primers and Whole Genome Sequencing Protocol

Frontiers in Medicine

George Githinji¹  D. James Nokes²  Arnold W. Lambisia³  Nickson Murunga³  Maureen W. Mburu³  Jane N. Mwangi³  Edidah O. Moraa³  Khadija S. Mohammed³  Leonard Ndwiga³  John M. Morobe³  Jennifer Musyoki³  Timothy O. Makori³  Charles N. Agoti⁴  Lynette Isabella Ochola-Oyier⁵ 

[1] Department of Biochemistry and Biotechnology, Pwani University, Kilifi, Kenya;Department of Biological Sciences, University of Warwick, Coventry, United Kingdom;Kenya Medical Research Institute (KEMRI)-Wellcome Trust Research Programme (KWTRP), Kilifi, Kenya;Nuffield Department of Medicine, Pwani University, Kilifi, Kenya;Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom;

关键词: ARTIC V4;    SARS-CoV-2;    whole genome sequencing;    amplicon drop-offs;    protocol;   

DOI  :  10.3389/fmed.2022.836728

来源: DOAJ

【 摘 要 】

IntroductionThe 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.MethodsWe 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'.ResultsWe 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.ConclusionUtilizing 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.

【 授权许可】

Unknown