2Department of Biological Sciences, University of Warwick, Coventry, United Kingdom. 1Kenya Medical Research Institute (KEMRI)-Wellcome Trust Research Programme (KWTRP), Kilifi, Kenya. Agoti 1,3, Lynette Isabella Ochola-Oyier 1,4 and George Githinji 1,5 Moraa 1, Jennifer Musyoki 1, Nickson Murunga 1, Jane N. I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.Arnold W. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Sample processing and analysis was covered by ethical approval for the use of anonymised oro- or nasopharyngeal specimens from patients for the diagnosis of influenza and other respiratory pathogens, including SARS-CoV-2 (North West-Greater Manchester South Research Ethics Committee, REC Ref:19/NW/0730). The details of the IRB/oversight body that provided approval or exemption for the research described are given below: I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. The views expressed are those of the author(s) and not necessarily those of the NHS, NIHR or the UKHSA. Computation used the Oxford Biomedical Research Computing (BMRC) facility, a joint development between the Wellcome Centre for Human Genetics and the Big Data Institute supported by Health Data Research UK and the NIHR Oxford Biomedical Research Centre. This study is supported by the National Institute for Health Research (NIHR) Health Protection Research Unit in Healthcare Associated Infections and Antimicrobial Resistance (NIHR200915), a partnership between the UK Health Security Agency (UKHSA) and the University of Oxford. Some Midnight sequencing kits used in this study were provided free of charge by Oxford Nanopore Technologies Funding Statement Approximate cost per sample for Midnight (£12) is lower than ARTIC (£16) while hands-on time is considerably lower for Midnight (∼7 hours) than ARTIC protocols (∼9.5 hours). 
Revised Midnight protocol recovered ≥90% complete genomes for 85.9% (61/71) of Omicron samples vs. The revised protocols both improved considerably the recovery of Omicron genomes and exhibited similar overall performance to one another. We evaluated Omicron sequencing performance using a revised Midnight primer mix alongside prototype ARTIC v4.1 primers, head-to-head with the existing commercially available Midnight and ARTIC v4 protocols.
Explaining these observations were ARTIC v4’s superior genome recovery in low viral titre/high cycle threshold (Ct) samples and inferior performance in high titre/low Ct samples, where Midnight excelled. Midnight protocol however yielded higher average genome recovery (mean 98.8%) than ARTIC v4 (98.1%) and ARTIC v3 (75.4%), resulting in less ambiguous final consensus assemblies overall. For Delta lineage samples, ARTIC v4 recovered the greatest proportion of ≥90% complete genomes (81.1% 159/193), followed by Midnight (71.5% 138/193) and ARTIC v3 (34.1% 14/41). We compared the performance of Oxford Nanopore Technologies (ONT) Midnight and ARTIC tiling amplicon protocols using 196 Delta lineage SARS-CoV-2 clinical specimens, and 71 mostly Omicron lineage samples with S gene target failure (SGTF), reflecting circulating lineages in the United Kingdom during December 2021.
Amplicon sequencing has proven very effective for sequencing SARS-CoV-2, but prevalent mutations disrupting primer binding sites have necessitated the revision of sequencing protocols in order to maintain performance for emerging virus lineages. Genome sequencing is pivotal to SARS-CoV-2 surveillance, elucidating the emergence and global dissemination of acquired genetic mutations.
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