Simplified point-of-care full SARS-CoV-2 genome sequencing using nanopore technology
Abstract
Background
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. Among available deep-sequencing technologies, nanopore-sequencing could be an important cornerstone, since it is mobile, scalable and costs-effective. Therefore, streamlined nanopore-sequencing protocols need to be developed and optimized for SARS-CoV-2 variants identification.
Results
We adapted and simplified existing workflows using the ‘midnight’ 1,200 bp amplicon split primer sets for PCR, which produce tiled overlapping amplicons covering almost the entire SARS-CoV-2 genome. Subsequently, we applied Oxford Nanopore Rapid Barcoding and the portable MinION Mk1C sequencer combined with the interARTIC bioinformatics pipeline. We tested a simplified and less time-consuming workflow using SARS-CoV-2-positive specimens from clinical routine and identified pre-analytical parameters, which may help to decrease sequencing failures rates. Complete pipeline duration was approx. 7 hrs for one specimen and approx. 11 hrs for 12 multiplexed barcoded specimens.
Conclusions
The adapted protocol contains less processing steps and can be completely conducted within one working-day. Diagnostic CT values are principal criteria for specimen selection.
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