{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,6]],"date-time":"2026-06-06T04:55:24Z","timestamp":1780721724927,"version":"3.54.1"},"update-to":[{"DOI":"10.12688\/wellcomeopenres.24735.1","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2026,4,21]],"date-time":"2026-04-21T00:00:00Z","timestamp":1776729600000}}],"reference-count":52,"publisher":"F1000 Research Ltd","license":[{"start":{"date-parts":[[2026,4,21]],"date-time":"2026-04-21T00:00:00Z","timestamp":1776729600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100010269","name":"Wellcome Trust","doi-asserted-by":"publisher","award":["206298"],"award-info":[{"award-number":["206298"]}],"id":[{"id":"10.13039\/100010269","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100010269","name":"Wellcome Trust Award","doi-asserted-by":"crossref","award":["313694\/Z\/24\/Z"],"award-info":[{"award-number":["313694\/Z\/24\/Z"]}],"id":[{"id":"10.13039\/100010269","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["wellcomeopenresearch.org"],"crossmark-restriction":false},"short-container-title":["Wellcome Open Res"],"abstract":"<ns5:p>Background The COVID-19 pandemic exposed critical vulnerabilities in global laboratory supply chains, disrupting the availability of key reagents and jeopardising the continuity of genomic surveillance for epidemic response. Sustaining sequencing capacity during shortages requires locally accessible alternatives to commercial kits. Methods We developed ARTIC HELP (Homebrew Enzymes for Library Preparation), an open-source adaptation of the ARTIC nanopore sequencing protocol for viral genomic surveillance. We described cost-effective, generic replacements for enzyme mixes used in tiling multiplex RT-PCR and the nanopore native barcoding workflow, including end-prep (EP), barcode ligation (BL), and adapter ligation (AL). Through systematic evaluation, we tested wild-type M-MLV reverse transcriptase and two types of proofreading DNA polymerases, (i) B-family Pfu-based polymerases fused to an Sso7d DNA-binding domain, and (ii) blends of A-family (Taq-based) and B-family (Pfu-based) polymerases, against standard reagents. We validated the workflow on clinical SARS-CoV-2 and Norovirus GII samples. Results The HELP workflow delivered genome coverage comparable to the ARTIC LoCost protocol. For SARS-CoV-2 samples (Ct \u226428), wild-type M-MLV RT combined with selected Pfu or AB blend polymerases, alongside optimized HELP EP, BL, and AL mixes, achieved 84.0\u201399.6% genome coverage. For Norovirus GII samples (Ct \u226432), the HELP workflow enabled &gt;85% coverage across six of eight genotypes tested. While some polymerases showed reduced performance at higher Ct values, they performed reliably at Ct &lt;24, supporting their use as emergency alternatives when viral load is high and RNA quality sufficient. Conclusions ARTIC HELP provides a practical and flexible solution to maintain viral sequencing capacity when standard ARTIC LoCost reagents are inaccessible or unaffordable. Our cost analysis highlights global disparities in reagent pricing, likely influenced by import fees, supply barriers, and local procurement conditions, underscoring the need for more equitable pricing models and local sourcing strategies. By expanding reagent options, ARTIC HELP strengthens preparedness for future global health emergencies.<\/ns5:p>","DOI":"10.12688\/wellcomeopenres.24735.2","type":"journal-article","created":{"date-parts":[[2026,4,21]],"date-time":"2026-04-21T10:49:10Z","timestamp":1776768550000},"page":"529","update-policy":"https:\/\/doi.org\/10.12688\/wellcomeopenres.crossmark-policy","source":"Crossref","is-referenced-by-count":1,"title":["Reducing Supply Chain Dependencies for Viral Genomic Surveillance: Get by with a Little HELP from Commercial Enzymes already in your Lab Freezer"],"prefix":"10.12688","volume":"10","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4929-0841","authenticated-orcid":false,"given":"Ganna","family":"Kovalenko","sequence":"first","affiliation":[{"name":"Division of Virology Cambridge England CB2 0QN University of Cambridge Department of Pathology"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3514-7681","authenticated-orcid":false,"given":"Myra","family":"Hosmillo","sequence":"additional","affiliation":[{"name":"Division of Virology Cambridge England CB2 0QN University of Cambridge Department of Pathology"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4269-0153","authenticated-orcid":false,"given":"Chris","family":"Kent","sequence":"additional","affiliation":[{"name":"Institute of Microbiology and Infection Birmingham England B15 2TT University of Birmingham School of Biosciences"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kess","family":"Rowe","sequence":"additional","affiliation":[{"name":"Division of Virology Cambridge England CB2 0QN University of Cambridge Department of Pathology"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andrew","family":"Rambaut","sequence":"additional","affiliation":[{"name":"Edinburgh Scotland EH1 2LE The University of Edinburgh Institute of Evolutionary Biology"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9843-8988","authenticated-orcid":false,"given":"Nicholas J","family":"Loman","sequence":"additional","affiliation":[{"name":"Institute of Microbiology and Infection Birmingham England B15 2TT University of Birmingham School of Biosciences"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joshua","family":"Quick","sequence":"additional","affiliation":[{"name":"Institute of Microbiology and Infection Birmingham England B15 2TT University of Birmingham School of Biosciences"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9483-510X","authenticated-orcid":false,"given":"Ian","family":"Goodfellow","sequence":"additional","affiliation":[{"name":"Division of Virology Cambridge England CB2 0QN University of Cambridge Department of Pathology"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"2560","published-online":{"date-parts":[[2026,4,21]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","first-page":"228-232","DOI":"10.1038\/nature16996","article-title":"Real-time, portable genome sequencing for Ebola surveillance.","volume":"530","author":"J Quick","year":"2016","journal-title":"Nature."},{"key":"ref2","doi-asserted-by":"publisher","first-page":"9-20","DOI":"10.1038\/nrg.2017.88","article-title":"Towards a genomics-informed, real-time, global pathogen surveillance system.","volume":"19","author":"J Gardy","year":"2018","journal-title":"Nat Rev Genet."},{"key":"ref3","doi-asserted-by":"publisher","first-page":"10-19","DOI":"10.1038\/s41564-018-0296-2","article-title":"Tracking virus outbreaks in the twenty-first century.","volume":"4","author":"N Grubaugh","year":"2019","journal-title":"Nat Microbiol."},{"key":"ref4","doi-asserted-by":"publisher","first-page":"146387","DOI":"10.1016\/j.gene.2022.146387","article-title":"Role of genomics in combating COVID-19 pandemic.","volume":"823","author":"K Saravanan","year":"2022","journal-title":"Gene."},{"key":"ref5","doi-asserted-by":"publisher","first-page":"105405","DOI":"10.1016\/j.meegid.2023.105405","article-title":"Global SARS-CoV-2 genomic surveillance: what we have learned (so far).","volume":"108","author":"S Tosta","year":"2023","journal-title":"Infect Genet Evol."},{"key":"ref6","article-title":"2022\u20132032. Geneva: World Health Organization","year":"2022"},{"key":"ref7","doi-asserted-by":"publisher","first-page":"406-410","DOI":"10.1038\/nature22401","article-title":"Establishment and cryptic transmission of Zika virus in Brazil and the Americas.","volume":"546","author":"N Faria","year":"2017","journal-title":"Nature."},{"key":"ref8","doi-asserted-by":"publisher","first-page":"mgen000991","DOI":"10.1099\/mgen.0.000991","article-title":"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.","volume":"9","author":"F Ulhuq","year":"2023","journal-title":"Microb Genom."},{"key":"ref9","doi-asserted-by":"publisher","first-page":"e48","DOI":"10.1590\/S1678-9946202264048","article-title":"Shotgun metagenomic sequencing of the first case of monkeypox virus in Brazil, 2022.","volume":"64","author":"I Claro","year":"2022","journal-title":"Rev Inst Med Trop Sao Paulo."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"598","DOI":"10.12688\/f1000research.53567.1","article-title":"Whole-genome sequencing of SARS-CoV-2 in Uganda: implementation of the low-cost ARTIC protocol in resource-limited settings [version 1; peer review: 2 approved].","volume":"10","author":"G Mboowa","year":"2021","journal-title":"F1000Res."},{"key":"ref11","doi-asserted-by":"publisher","first-page":"15749","DOI":"10.1038\/s41598-022-19414-y","article-title":"Tracking SARS-COV-2 variants using Nanopore sequencing in Ukraine in 2021.","volume":"12","author":"A Yakovleva","year":"2022","journal-title":"Sci Rep."},{"key":"ref12","doi-asserted-by":"publisher","first-page":"e0001455","DOI":"10.1371\/journal.pgph.0001455","article-title":"Implementation of genomic surveillance of SARS-CoV-2 in the Caribbean: lessons learned for sustainability in resource-limited settings.","volume":"3","author":"N Sahadeo","year":"2023","journal-title":"PLOS Glob Public Health."},{"key":"ref13","doi-asserted-by":"publisher","DOI":"10.1101\/2024.12.20.629611","article-title":"PrimalScheme: open-source community resources for low-cost viral genome sequencing.","author":"C Kent","year":"2024","journal-title":"bioRxiv."},{"key":"ref14","doi-asserted-by":"publisher","DOI":"10.17504\/protocols.io.bp2l6n26rgqe\/v3","article-title":"nCoV-2019 sequencing protocol v3 (LoCost) V.3.","author":"J Quick","year":"2020","journal-title":"protocols.io."},{"key":"ref15","doi-asserted-by":"publisher","DOI":"10.1101\/2020.09.04.283077","article-title":"Improvements to the ARTIC multiplex PCR method for SARS-CoV-2 genome sequencing using nanopore.","author":"J Tyson","year":"2020","journal-title":"bioRxiv."},{"key":"ref16","doi-asserted-by":"publisher","first-page":"20273","DOI":"10.1038\/s41598-024-70416-4","article-title":"Reconstructing SARS-CoV-2 lineages from mixed wastewater sequencing data.","volume":"14","author":"I Ellmen","year":"2024","journal-title":"Sci Rep."},{"key":"ref17","doi-asserted-by":"publisher","first-page":"102983","DOI":"10.1016\/j.ijdrr.2022.102983","article-title":"Supply chain disruption during the COVID-19 pandemic: recognizing potential disruption management strategies.","volume":"75","author":"J Moosavi","year":"2022","journal-title":"Int J Disaster Risk Reduct."},{"key":"ref18","doi-asserted-by":"publisher","first-page":"1146730","DOI":"10.3389\/fpubh.2023.1146730","article-title":"Strengthening pathogen genomic surveillance for health emergencies: insights from the World Health Organization's regional initiatives.","volume":"11","author":"O Akande","year":"2023","journal-title":"Front Public Health."},{"key":"ref19","doi-asserted-by":"publisher","first-page":"e00215-e00220","DOI":"10.1128\/mBio.00215\u201320","article-title":"Norovirus replication in human intestinal epithelial cells is restricted by the interferon-induced JAK\/STAT signaling pathway and RNA polymerase II-mediated transcriptional responses.","volume":"11","author":"M Hosmillo","year":"2020","journal-title":"mBio."},{"key":"ref20","doi-asserted-by":"publisher","first-page":"2000045","DOI":"10.2807\/1560-7917.ES.2020.25.3.2000045","article-title":"Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR.","volume":"25","author":"V Corman","year":"2020","journal-title":"Euro Surveill."},{"key":"ref21","doi-asserted-by":"publisher","first-page":"245","DOI":"10.12688\/wellcomeopenres.17078.1","article-title":"Design, development, and validation of a strand-specific RT-qPCR assay for GI and GII human noroviruses [version 1; peer review: 3 approved].","volume":"6","author":"K K\u00f6nig","year":"2021","journal-title":"Wellcome Open Res."},{"key":"ref22","article-title":"SARS-CoV-2 v4.1.0 primer scheme, primalscheme labs","year":"n.d."},{"key":"ref23","article-title":"Norovirus pan-GII v1.1.0 primer scheme. primalscheme labs","year":"n.d."},{"key":"ref24","article-title":"2019 novel coronavirus bioinformatics protocol.","author":"N Loman","year":"2020","journal-title":"ARTIC Network"},{"key":"ref25","article-title":"ncov-tools: Small collection of tools for performing quality control on coronavirus sequencing data and genomes. [Software] GitHub","author":"J Simpson","year":"n.d."},{"key":"ref26","article-title":"The ARTIC field bioinformatics pipeline. [Software]. GitHub","year":"n.d."},{"key":"ref27","article-title":"RAMPART: Read assignment, mapping, and phylogenetic analysis in real time. [Software]. GitHub","year":"n.d."},{"key":"ref28","doi-asserted-by":"publisher","first-page":"121-125","DOI":"10.1016\/j.jcv.2011.03.006","article-title":"An automated genotyping tool for enteroviruses and noroviruses.","volume":"51","author":"A Kroneman","year":"2011","journal-title":"J Clin Virol."},{"key":"ref29","doi-asserted-by":"publisher","first-page":"3","DOI":"10.12688\/wellcomeopenres.15518.2","article-title":"Rapid in-country sequencing of whole virus genomes to inform rabies elimination programmes [version 2; peer review: 3 approved].","volume":"5","author":"K Brunker","year":"2020","journal-title":"Wellcome Open Res."},{"key":"ref30","doi-asserted-by":"publisher","first-page":"24","DOI":"10.1186\/s12985-020-1294-6","article-title":"Assessment of a multiplex PCR and Nanopore-based method for dengue virus sequencing in Indonesia.","volume":"17","author":"S Stubbs","year":"2020","journal-title":"Virol J."},{"key":"ref31","doi-asserted-by":"publisher","first-page":"mgen000920","DOI":"10.1099\/mgen.0.000920","article-title":"Multiplex MinION sequencing suggests enteric adenovirus F41 genetic diversity comparable to pre-COVID-19 era.","volume":"9","author":"M Maes","year":"2023","journal-title":"Microb Genom."},{"key":"ref32","doi-asserted-by":"publisher","first-page":"446","DOI":"10.1186\/s12879-023-08423-5","article-title":"Hepatitis C Virus in people with experience of injection drug use following their displacement to Southern Ukraine before 2020.","volume":"23","author":"A Yakovleva","year":"2023","journal-title":"BMC Infect Dis."},{"key":"ref33","doi-asserted-by":"publisher","first-page":"114-120","DOI":"10.7171\/jbt.21-3203-006","article-title":"Homebrew reagents for low-cost RT-LAMP.","volume":"32","author":"T Matute","year":"2021","journal-title":"J Biomol Tech."},{"key":"ref34","doi-asserted-by":"publisher","first-page":"100186","DOI":"10.1016\/j.crmeth.2022.100186","article-title":"Homebrew: an economical and sensitive glassmilk-based nucleic-acid extraction method for SARS-CoV-2 diagnostics.","volume":"2","author":"R Page","year":"2022","journal-title":"Cell Rep Methods."},{"key":"ref35","doi-asserted-by":"publisher","first-page":"410-419","DOI":"10.1111\/2041-210X.12871","article-title":"Single-tube library preparation for degraded DNA.","volume":"9","author":"C Car\u00f8e","year":"2018","journal-title":"Methods Ecol Evol."},{"key":"ref36","doi-asserted-by":"publisher","DOI":"10.17504\/protocols.io.dm6gp33w5vzp\/v2","article-title":"Economical Nanopore LSK sequencing: adapted protocols with homebrew reagents V.2.","author":"J Ou","year":"2024","journal-title":"protocols.io."},{"key":"ref37","doi-asserted-by":"publisher","first-page":"e48616","DOI":"10.1371\/journal.pone.0048616","article-title":"Library preparation and multiplex capture for massive parallel sequencing applications made efficient and easy.","volume":"7","author":"M Neiman","year":"2012","journal-title":"PLoS One."},{"key":"ref38","doi-asserted-by":"publisher","first-page":"6408-6411","DOI":"10.1039\/c8cc02414f","article-title":"XNA ligation using T4 DNA ligase in crowding conditions.","volume":"54","author":"D Kestemont","year":"2018","journal-title":"Chem Commun (Camb)."},{"key":"ref39","doi-asserted-by":"publisher","first-page":"7853-7871","DOI":"10.1093\/nar\/11.22.7853","article-title":"Polymer-stimulated ligation: enhanced blunt- or cohesive-end ligation of DNA or deoxyribooligonucleotides by T4 DNA ligase in polymer solutions.","volume":"11","author":"B Pheiffer","year":"1983","journal-title":"Nucleic Acids Res."},{"key":"ref40","doi-asserted-by":"publisher","first-page":"1997-2008","DOI":"10.1093\/nar\/13.6.1997","article-title":"Hexamine cobalt chloride promotes intermolecular ligation of blunt end DNA fragments by T4 DNA ligase.","volume":"13","author":"J Rusche","year":"1985","journal-title":"Nucleic Acids Res."},{"key":"ref41","doi-asserted-by":"publisher","first-page":"129-133","DOI":"10.1007\/BF02686107","article-title":"Effects of additives on efficiency and specificity of ligase detection reaction.","volume":"35","author":"Z Xiao","year":"2007","journal-title":"Mol Biotechnol."},{"key":"ref42","doi-asserted-by":"publisher","first-page":"6315-6327","DOI":"10.1016\/j.csbj.2021.11.030","article-title":"M-MuLV reverse transcriptase: selected properties and improved mutants.","volume":"19","author":"I Oscorbin","year":"2021","journal-title":"Comput Struct Biotechnol J."},{"key":"ref43","doi-asserted-by":"publisher","first-page":"61-77","DOI":"10.1007\/BF02762340","article-title":"Reverse transcriptase. The use of cloned moloney murine leukemia virus reverse transcriptase to synthesize DNA from RNA.","volume":"8","author":"G Gerard","year":"1997","journal-title":"Mol Biotechnol."},{"key":"ref44","doi-asserted-by":"publisher","first-page":"e202200521","DOI":"10.1002\/cbic.202200521","article-title":"Reverse transcriptases: from discovery and applications to Xenobiology.","volume":"24","author":"L Huber","year":"2023","journal-title":"Chembiochem."},{"key":"ref45","doi-asserted-by":"publisher","first-page":"1261-1276","DOI":"10.1038\/nprot.2017.066","article-title":"Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples.","volume":"12","author":"J Quick","year":"2017","journal-title":"Nat Protoc."},{"key":"ref46","doi-asserted-by":"publisher","first-page":"836728","DOI":"10.3389\/fmed.2022.836728","article-title":"Optimization of the SARS-CoV-2 ARTIC network V4 primers and whole genome sequencing protocol.","volume":"9","author":"A Lambisia","year":"2022","journal-title":"Front Med."},{"key":"ref47","doi-asserted-by":"publisher","first-page":"1393-1406","DOI":"10.1099\/jgv.0.001318","article-title":"Updated classification of norovirus genogroups and genotypes.","volume":"100","author":"P Chhabra","year":"2019","journal-title":"J Gen Virol."},{"key":"ref48","doi-asserted-by":"publisher","first-page":"e0216522","DOI":"10.1128\/aem.02165-22","article-title":"Amplicon-based high-throughput sequencing method for genotypic characterization of norovirus in oysters.","volume":"89","author":"A Fitzpatrick","year":"2023","journal-title":"Appl Environ Microbiol."},{"key":"ref49","doi-asserted-by":"publisher","first-page":"972-975","DOI":"10.1136\/jech-2022-219333","article-title":"Global inequity of COVID-19 diagnostics: challenges and opportunities.","volume":"76","author":"S Narayanasamy","year":"2022","journal-title":"J Epidemiol Community Health."},{"key":"ref50","doi-asserted-by":"publisher","DOI":"10.1101\/2025.06.11.658579","article-title":"Reducing supply chain dependencies for viral genomic surveillance: get by with a little HELP from commercial enzymes already in your lab freezer.","author":"G Kovalenko","year":"2025","journal-title":"bioRxiv."},{"key":"ref51","article-title":"Reducing supply chain dependencies for viral sequencing using ARTIC-HELP workflows.","author":"G Kovalenko","year":"2025","journal-title":"NCBI SRA."},{"key":"ref53","doi-asserted-by":"publisher","DOI":"10.17504\/protocols.io.36wgqqzq5gk5\/v1","article-title":"ARTIC HELP protocol for amplicon-based viral genome sequencing.","author":"G Kovalenko","year":"2025"}],"container-title":["Wellcome Open Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-529\/v2\/xml","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-529\/v2\/pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-529\/v2\/iparadigms","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,22]],"date-time":"2026-05-22T04:09:21Z","timestamp":1779422961000},"score":1,"resource":{"primary":{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-529\/v2"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,4,21]]},"references-count":52,"URL":"https:\/\/doi.org\/10.12688\/wellcomeopenres.24735.2","relation":{"has-version":[{"id-type":"doi","id":"10.12688\/wellcomeopenres.24735.1","asserted-by":"subject"}],"has-review":[{"id-type":"doi","id":"10.21956\/wellcomeopenres.29150.r155591","asserted-by":"subject"},{"id-type":"doi","id":"10.21956\/wellcomeopenres.29150.r155591","asserted-by":"object"}]},"ISSN":["2398-502X"],"issn-type":[{"value":"2398-502X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,4,21]]},"assertion":[{"value":"Indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-529\/v2#article-reports","order":0,"name":"referee-status","label":"Referee status","group":{"name":"current-referee-status","label":"Current Referee Status"}},{"value":"10.21956\/wellcomeopenres.27252.r135094, Leonardo C. Caserta, Cornell University, Ithaca, NY, 22 Nov 2025, version 1, 2 approved with reservations","URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-529\/v1#referee-response-135094","order":0,"name":"referee-response-135094","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Ganna Kovalenko<\/b>; \n<i>Posted: 11 Apr 2026<\/i>; We sincerely appreciate the reviewer\u2019s positive evaluation and helpful suggestion, which helped us further improve the manuscript.  \n<b>Reviewer Comment:<\/b> The study provides valuable insights into the flexibility of tiled amplicon whole-genome sequencing protocols, offering alternative and cost-effective reagents for library preparation. One important metric, however, was not included: the average read length per sample (or per workflow). Amplicon sizes can vary significantly depending on the enzymes used, which in turn affects the number of reads generated. This variability is especially pronounced when using long-read sequencing technologies. If this metric is not included in the analysis, it should at least be acknowledged as a limitation of the study. \n<b>Response: <\/b>We sincerely thank the reviewer for this helpful suggestion. The median read length metric was already included in the sequencing quality control metrics reported in the Extended data Tables S4\u2013S6 but was not mentioned in the main text. Following this comment, \n<b>we have now clearly highlighted this metric in the main text to make it easier for readers to identify. <\/b> \n<i>(lines 202-209):<\/i>\n<b> <\/b>\n<i>Performance metrics included amplicon post-PCR concentration, read count, N50, median read length, number and percentage of mapped reads, mean and average depth, genome coverage breadth, and amplicon dropouts (regions with \u226420&times; depth), detailed in Extended data Table S4 (Stages 1 and 2), Table S5 (Stage 3 SARS-CoV-2), and Table S6 (Stage 3 Norovirus GII). Across all experimental stages, median read lengths were highly consistent between ARTIC LoCost and HELP workflows (~525 bp for SARS-CoV-2 and ~1 kb for Norovirus GII), demonstrating that the use of alternative polymerases and library preparation reagents in the HELP protocol does not alter amplicon size distribution.<\/i>","URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-529\/v2#referee-comment-6508","order":1,"name":"referee-comment-6508","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.21956\/wellcomeopenres.27252.r141594, Tibutius Thanesh Pramanayagam Jayadas, University of Sri Jayewardenepura, Nugegoda, 19 Dec 2025, version 1, 2 approved with reservations","URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-529\/v1#referee-response-141594","order":2,"name":"referee-response-141594","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Ganna Kovalenko<\/b>; \n<i>Posted: 11 Apr 2026<\/i>; We sincerely thank the reviewer for the thoughtful and constructive feedback, which helped us improve the clarity of the manuscript.  \n<b>Reviewer Comment 1:<\/b>&nbsp; Core conditions are described (RT temperatures, PCR cycling, library prep conceptual composition, sequencing and bioinformatics overview).&nbsp;&nbsp;The authors also provide extended data and protocols via GitHub\/protocols.io, which is positive.&nbsp;However, for replication from the manuscript alone, several critical implementation details are either externalized or not fully specified: Replication-critical details to bring into the paper (must address): \n<b>1)<\/b> Replicates and randomization: For each workflow\/condition state the number of independent replicates and whether comparisons were run on the same flow cell or across multiple runs (this strongly affects depth and dropout comparisons).&nbsp;  \n<b>Response: <\/b>We thank the reviewer for these helpful comments and for highlighting important points to improve our study. The experimental design was organised into three sequential stages, each performed on a separate flow cell. Within each stage, workflows were evaluated by preparing separate libraries from the same amplicon pools, enabling direct comparison of workflows under the same experimental conditions. Libraries prepared with different adapter ligation (AL) mixes were sequenced on separate flow cells, as Oxford Nanopore library preparation protocols require libraries loaded on the same flow cell to use the same adapter mix. \n<b>We have now clarified this in the Methods section:<\/b> \n<i>(lines 123-125): Within each stage, workflows were compared by preparing separate libraries from the same amplicon pools under identical experimental conditions.<\/i> \n<i>(lines 175-177): Experiments were organised into sequential stages, with each stage sequenced on a new flow cell. Libraries prepared with different adapter ligation mixes were sequenced on separate flow cells following ONT library preparation requirements.<\/i>  \n<b>2)<\/b> Input normalization: Clarify whether amplicon inputs into EP\/BL\/AL were normalized to the same ng amount across workflows and samples (you mention quantification specify normalization strategy).&nbsp;Exact barcoding\/ligation stoichiometry: You describe barcode dilution and volumes; add the final molar or mass assumptions. \n<b>Response: <\/b>We appreciate the reviewer's helpful suggestions regarding input normalization and reaction stoichiometry. Amplicon pools were quantified after PCR cleanup as a quality control step to assess amplicon yield and suitability for library preparation. However, library preparation followed the ARTIC LoCost workflow, which uses fixed reaction volumes rather than normalization to a defined DNA mass or molar input. As HELP was designed as a drop-in alternative to the ARTIC LoCost protocol, we followed the same volume-based reaction setup for end-prep, barcoding, and adapter ligation steps. This approach is widely used and has been extensively validated in nanopore-based viral genome sequencing\n<b>.  We have now clarified this in the Methods section<\/b>: \n<i>(lines 125\u2013127): End-prep, barcoding and adapter ligation reactions were performed using fixed reaction volumes according to the ARTIC LoCost protocol rather than molar normalization of DNA input.<\/i>   \n<b>3)<\/b> Computational reproducibility: Provide versions\/commit hashes for ARTIC pipeline, medaka model version, ncov-tools version and basecalling model details beyond \u201chigh accuracy model,\u201d because these change consensus outcomes.&nbsp;  \n<b>Response: <\/b>We appreciate the reviewer's comments. To improve computational reproducibility, we have now specified the versions of the ARTIC pipeline, medaka, and ncov-tools (\n<i>lines 179-185<\/i>), as well as the basecalling model configuration (dna_r9.4.1_e8_hac@v3.3) used (\n<i>line 175<\/i>). These details have been added to the Methods section of the revised manuscript. &nbsp; \n<b>Reviewer Comment 2:<\/b> \n<b>If applicable, is the statistical analysis and its interpretation appropriate?<\/b> For a methods\/workflow paper, descriptive metrics and visualisations are acceptable. &nbsp; However, the manuscript repeatedly compares workflows (sometimes with small differences) without quantifying uncertainty, effect sizes or reproducibility. There are also multiple conditions (polymerases, mixes, Ct strata) which is exactly where minimal statistics would strengthen interpretability.  \n<b>Response:<\/b> We appreciate this important suggestion. We have revised the Results section to strengthen interpretability by incorporating quantitative comparisons across workflows and conditions. Specifically, we now report genome coverage ranges across Ct strata in Stages 1-3 and refine comparisons across polymerases and library preparation steps to better illustrate effect sizes and variability. We hope these additions provide clearer and more transparent comparisons of workflow performance. &nbsp;  \n<b>Reviewer Comment 3:<\/b> \n<b>Are the conclusions drawn adequately supported by the results?<\/b> The central operational conclusion HELP can achieve ARTIC-like coverage for many samples, especially at lower Ct \/ higher viral load and can maintain sequencing continuity during shortages is supported by the presented coverage and dropout data.&nbsp;\n<u>Where conclusions drift beyond the data:<\/u> \n<b>1)<\/b> Claims about general robustness may be too broad given limited external validation (single lab setting, limited number of enzymes tested, limited replication described).&nbsp; \n<b>Response:<\/b> We appreciate the reviewer raising this point. Our intention was to describe robustness in the context of a drop-in alternative to the ARTIC LoCost workflow, where experimental conditions were kept consistent to enable direct comparison. We agree that the term should be interpreted within the scope of the conditions evaluated in this study, however we recognise that validation of the approach requires the user community to further validate the protocol, beyond the scope of the current study.  \n<b>We have therefore revised the manuscript to clarify that HELP demonstrated robust performance under the conditions tested<\/b>. In the Abstract: instead of&nbsp; \u201c\n<i>ARTIC HELP provides a robust and flexible solution to maintain viral sequencing capacity when standard reagents are inaccessible or unaffordable<\/i>\u201d we now use the following wording \u201c\n<i>ARTIC HELP provides a practical and flexible solution to maintain viral sequencing capacity when standard ARTIC LoCost reagents are inaccessible or unaffordable<\/i>\u201d. (Conclusion; \n<i>lines 603-604<\/i>) Instead of \u201c\n<i>We developed ARTIC HELP, a robust workflow using alternative enzymes and home-brew buffers that works as well as the standard ARTIC LoCost protocol for sequencing viral genomes from clinical samples<\/i>\u201d we now use the following wording \u201c\n<i>We developed ARTIC HELP, a workflow using alternative enzymes and home-brew buffers that performed comparably to the standard ARTIC LoCost protocol for sequencing viral genomes from clinical samples<\/i>.\u201d  \n<b>2)<\/b> The cost inequity discussion is important but the inference that disparities are driven \u201crather than protocol complexity\u201d is not fully demonstrated complexity is not quantitatively measured and procurement ecosystems differ. The table shows price variation the causal attribution should be softened or better supported.&nbsp;  \n<b>Response:<\/b> We appreciate this important point. We agree that our analysis shows cross-country variation in reagent pricing but does not directly quantify the contribution of importation protocol complexity. We have therefore revised the manuscript to soften the causal interpretation and to state more cautiously that the observed disparities are likely influenced by import fees, shipping costs, taxes, and local procurement conditions. (Abstract) : Instead of \n<i>\u201c..driven by import fees and supply barriers, rather than protocol complexity..<\/i>\u201d we now use the following wording \n<i>\u201c.. likely influenced by import fees, supply barriers, and local procurement conditions.<\/i>.\u201d (Discussion, \n<i>lines: 578-581<\/i>) Instead of \n<i>\u201c..with these cost increases arising not due to technical differences in the supplied products, but largely to customs fees, high import taxes, and shipping costs.<\/i>\u201d we now use the following wording \n<i>\u201c..with these cost differences likely reflecting a combination of customs fees, import taxes, shipping costs, and local procurement conditions<\/i>.\u201d   \n<b>3)<\/b> Norovirus conclusions should be framed carefully: the data strongly suggest genotype\/primer-scheme effects dominate coverage for some genotypes but additional experiments (primer mismatch analysis, rebalancing, alternate schemes) would be needed before recommending HELP broadly for \u201cNorovirus GII\u201d without caveats.&nbsp;  \n<b>Response:<\/b> We are thankful for this constructive suggestion. We agree that the norovirus findings should be interpreted more cautiously. We have revised the manuscript to clarify that the results demonstrate the applicability of the HELP workflow for the genotypes tested, while also highlighting that performance was influenced by genotype-specific primer-scheme effects. We also adjusted the Discussion and Limitation sections. (Results section, \n<i>lines 426-429<\/i>) Instead of \u201c\n<i>These results demonstrated that the HELP workflow is robust and adaptable for sequencing Norovirus genogroup II, confirming its applicability beyond SARS-CoV-2<\/i>.\u201d we now use the following wording&nbsp; \u201c\n<i>These results indicate that the HELP workflow can be applied to Norovirus GII for the genotypes tested in this study, although performance was influenced by genotype-specific primer-scheme effects.<\/i>\u201d (Limitation section, \n<i>lines 597-601<\/i>) instead of \u201c\n<i>Although the current norovirus scheme is still under development, our findings provide insights for further optimization<\/i>\u201d, we now use the following wording \u201c\n<i>As the current norovirus scheme is still under development, our findings should be interpreted as supporting initial use for the genotypes tested, while highlighting the need for further optimization<\/i>.\u201d","URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-529\/v2#referee-comment-6509","order":3,"name":"referee-comment-6509","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.21956\/wellcomeopenres.29150.r155591, Aloysious Ssemaganda, Central Public Health Laboratories, Kampala, 22 May 2026, version 2, indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-529\/v2#referee-response-155591","order":4,"name":"referee-response-155591","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"This work was supported by Wellcome [206298] [313694]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.","order":5,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.","order":0,"name":"copyright-info","label":"Copyright"}]}}