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. 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.
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