![]() ![]() Moreover, the search for the zoonotic origin of SARS-CoV-2 from comparative analyses of genomic data is an ongoing issue with relevance for the early recognition of future outbreak scenarios. A particular focus is given to the identification of VOCs with accelerated transmission rates, increased infectivity or immune escape mutations as these variants would warrant adaptations in containment and vaccination strategies. To face the ongoing SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) pandemic, a global decentralized warning system is being established to recognize local outbreaks and the emergence of novel variants of concern (VOC). Thus, based on the described thermocycler/chemistry combination, we recommend CT values of ~26 or lower to achieve full and high-quality SARS-CoV-2 (+)RNA genome coverage. As a guideline, SARS-CoV-2 genome copy numbers lower than 4 × 10 6 were associated with a coverage threshold below 20-fold and incompletely assembled SARS-CoV-2 genomes. Diagnostic CT values deduced from qPCR standardization experiments can act as principal criteria for specimen selection. ![]() The adapted protocol contains fewer processing steps and can be completely conducted within one working day. 11 h for 12 multiplexed barcoded specimens. We tested a simplified and less time-consuming workflow using SARS-CoV-2-positive specimens from clinical routine and identified the CT value as a useful pre-analytical parameter, which may help to decrease sequencing failures rates. Subsequently, we applied Oxford Nanopore Rapid Barcoding and the portable MinION Mk1C sequencer combined with the interARTIC bioinformatics pipeline. We adapted and simplified existing workflows using the ‘midnight’ 1200 bp amplicon split primer sets for PCR, which produce tiled overlapping amplicons covering almost the entire SARS-CoV-2 genome. Therefore, streamlined nanopore-sequencing protocols need to be developed and optimized for SARS-CoV-2 variants identification. Among available deep-sequencing technologies, nanopore-sequencing could be an important cornerstone, as it is mobile, scalable, and cost-effective. "Nanopore sequencing has been the workhorse for us generating data as we looked at how the virus was spreading differently through different parts of our state.The scale of the ongoing SARS-CoV-2 pandemic warrants the urgent establishment of a global decentralized surveillance system to recognize local outbreaks and the emergence of novel variants of concern. Katarina Braun, University of Wisconsin has been using nanopore sequencing throughout the pandemic. "Our capacity to run sequencing of SARS-CoV-2 has gone on to inform some of the resulting control measures." George Githinji from KEMRI, Wellcome Trust has been able to establish nanopore sequencing for COVID-19 surveillance in Kenya: It also provides scientists with information about how strains of the virus are related, helping to indicate routes of transmission and investigate clusters - all ultimately helping to inform strategies to control the spread. Rapid sequencing and sharing of SARS-CoV-2 genomic data enables rapid identification of variants and effective tracking of their prevalence and distribution. Making use of the Rapid Sequencing Kits, the approach has the fastest turnaround time, least hands-on time, and lowest cost-per-sample it is also a good choice for those wishing to incorporate automation. The recently released Midnight protocol is optimised for high throughput requirements, for highly multiplexed SARS-CoV-2 genome sequencing. The protocol is best for those with some prior experience of nanopore sequencing. Featuring a normalisation step, the method is ideal for the routine sequencing of smaller batches of samples spanning a wide range of Ct values, and is optimised for maximum coverage across all genomes sequenced. The ARTIC Classic protocol was the first SARS-CoV-2 nanopore sequencing protocol to be utilised, and is the most well established. Oxford Nanopore offers a number of protocols for sequencing SARS-CoV-2, the most utilised being the ARTIC Classic and the Midnight protocols. ![]() Research teams are able to access sequencing technology in days, without capital cost, for quick set-up of rapid turnaround SARS-CoV-2 sequencing.įrom initial characterisation of the SARS-CoV-2 virus genome to the rapid identification of variants, researchers have been utilising nanopore sequencing to generate data essential to combating the spread of COVID-19. Oxford Nanopore has released new protocols enabling the rapid, cost effective sequencing of SARS-CoV-2 - the virus that causes COVID-19. ![]()
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