{"doi":"10.3389/fmed.2022.836728","title":"Optimization of the SARS-CoV-2 ARTIC Network V4 Primers and Whole Genome Sequencing Protocol","abstract":"<jats:sec><jats:title>Introduction</jats:title><jats:p>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.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>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'.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>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% (<jats:italic>n</jats:italic> = 31) of the samples. However, only 60–70% of the genomes could be recovered in samples that had &amp;lt;28% genome coverage with the ARTIC V3 primers. There was no statistically significant (<jats:italic>p</jats:italic> &amp;gt; 0.05) correlation between Ct value and genome recovery.</jats:p></jats:sec><jats:sec><jats:title>Conclusion</jats:title><jats:p>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.</jats:p></jats:sec>","journal":"Frontiers in Medicine","year":2022,"id":617502,"datarank":3.0060721104064188,"base_score":4.477336814478207,"endowment":4.477336814478207,"self_citation_contribution":0.6716005221717312,"citation_network_contribution":2.3344715882346874,"self_endowment_contribution":0.6716005221717312,"citer_contribution":2.3344715882346874,"corpus_percentile":null,"corpus_rank":null,"citation_count":87,"citer_count":79,"citers_with_citation_signal":59,"citers_with_endowment":59,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":null,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":null,"fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1592522,"name":"Khadija S. Mohammed","orcid":null,"position":1,"is_corresponding":false},{"id":1592527,"name":"Timothy O. Makori","orcid":null,"position":2,"is_corresponding":false},{"id":905186,"name":"Leonard Ndwiga","orcid":"0000-0002-2936-1059","position":3,"is_corresponding":false},{"id":1592531,"name":"Maureen W. Mburu","orcid":null,"position":4,"is_corresponding":false},{"id":1592533,"name":"John M. Morobe","orcid":null,"position":5,"is_corresponding":false},{"id":1592534,"name":"Edidah O. Moraa","orcid":null,"position":6,"is_corresponding":false},{"id":746407,"name":"Jennifer Musyoki","orcid":"0000-0001-7002-9384","position":7,"is_corresponding":false},{"id":1020127,"name":"Nickson Murunga","orcid":"0000-0001-5304-2210","position":8,"is_corresponding":false},{"id":1592535,"name":"Jane N. Mwangi","orcid":null,"position":9,"is_corresponding":false},{"id":621551,"name":"D. James Nokes","orcid":"0000-0001-5426-1984","position":10,"is_corresponding":false},{"id":621548,"name":"Charles N. 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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.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>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'.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>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. 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There was no statistically significant (<jats:italic>p</jats:italic> &amp;gt; 0.05) correlation between Ct value and genome recovery.</jats:p></jats:sec><jats:sec><jats:title>Conclusion</jats:title><jats:p>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.</jats:p></jats:sec>","is_dataset_classified":null,"base_score":4.477336814478207,"endowment":4.477336814478207,"datacite_reuse_total":0,"file_count":0,"downloads":0,"views":0,"has_version_chain":false,"is_dataset":false,"is_oa":false,"pmid":"35252269","pmcid":"PMC8891481","openalex_id":"https://openalex.org/W4213162735","authors":[],"funders":[{"funder_name":"Wellcome","grant_id":"102975","title":"Defining pathways of respiratory virus transmission leading to improved intervention strategies."},{"funder_name":"Wellcome","grant_id":"107769/Z/10/Z","title":null},{"funder_name":"Wellcome","grant_id":"220985","title":"COVID-19 Intervention Modelling for East Africa (CIMEA)"},{"funder_name":"National Institute for Health Research (NIHR)","grant_id":"17/63/82","title":null},{"funder_name":"Wellcome Trust","grant_id":"102975/Z/13/Z","title":null},{"funder_name":"Wellcome Trust","grant_id":"107568/Z/15/Z","title":null},{"funder_name":"National Institute for Health Research (NIHR)","grant_id":"16/136/33","title":null},{"funder_name":"Wellcome Trust","grant_id":"107769","title":"Initiative to Develop African Research Leaders (IDeAL)"},{"funder_name":"Wellcome Trust","grant_id":"unidentified","title":"unidentified"},{"funder_name":"Wellcome Trust","grant_id":"","title":null},{"funder_name":"Wellcome Trust","grant_id":"","title":null}],"total_grants":11,"fwci":7.9219,"citation_percentile":0.98368298,"influential_citations":0,"citation_trend":[{"year":2021,"count":1},{"year":2022,"count":16},{"year":2023,"count":33},{"year":2024,"count":22},{"year":2025,"count":11},{"year":2026,"count":4}],"oa_status":"gold","license":"cc-by","oa_locations":[{"url":"https://doi.org/10.3389/fmed.2022.836728","host_type":"journal"},{"url":"https://doi.org/10.3389/fmed.2022.836728","host_type":"publisher"},{"url":"https://www.frontiersin.org/articles/10.3389/fmed.2022.836728/full","host_type":"publisher"},{"url":"https://pubmed.ncbi.nlm.nih.gov/35252269","host_type":"repository"},{"url":"https://doaj.org/article/aae8a08ade2e449fa58177b8d0b5e14d","host_type":"repository"},{"url":"https://ora.ox.ac.uk/objects/uuid:c9678c34-3afd-4f01-b8ef-07cc5d96dc56","host_type":"repository"},{"url":"https://www.ncbi.nlm.nih.gov/pmc/articles/8891481","host_type":"repository"},{"url":"https://europepmc.org/articles/PMC8891481","host_type":"Europe_PMC"},{"url":"https://europepmc.org/articles/PMC8891481?pdf=render","host_type":"Europe_PMC"},{"url":"https://www.frontiersin.org/articles/10.3389/fmed.2022.836728/pdf","host_type":""},{"url":"https://dx.doi.org/10.60692/nyg19-x9s23","host_type":""},{"url":"https://dx.doi.org/10.60692/z32rq-qjm12","host_type":""},{"url":"http://dx.doi.org/10.3389/fmed.2022.836728","host_type":""},{"url":"http://wrap.warwick.ac.uk/163387/7/WRAP-optimization-SARS-CoV-2-ARTIC-network-V4-primers-whole-genome-sequencing-protocol-Nokes-2022.pdf","host_type":""}],"fields_of_study":["SARS-CoV-2 and COVID-19 Research","SARS-CoV-2 detection and testing","Animal Virus Infections Studies","0301 basic medicine","03 medical and health sciences","0303 health sciences"],"mesh_terms":[],"keywords":["Amplicon","Primer (cosmetics)","Pooling","Computational biology","Protocol (science)","Genome","Computer science","Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)","Nat","Biology","Coronavirus disease 2019 (COVID-19)","Genetics","Computer network","Gene","Artificial intelligence","Polymerase chain reaction","Medicine","Chemistry","Protocol","Whole Genome Sequencing","Sars-cov-2","Amplicon Drop-Offs","Artic V4","Medicine (General)","Deep sequencing","Organic chemistry","Coronavirus Disease 2019 Research","FOS: Health sciences","Diagnostic Methods for COVID-19 Detection","Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins","R5-920","Biochemistry, Genetics and Molecular Biology","Health Sciences","Molecular Biology","QH","Life Sciences","Multiplex Genome Editing","Infectious Diseases","FOS: Biological sciences","RA"],"sdg_mappings":[{"sdg_number":3,"sdg_label":"3. 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