RESUMO
BackgroundThe 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. ResultsWe 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. ConclusionsThe adapted protocol contains less processing steps and can be completely conducted within one working-day. Diagnostic CT values are principal criteria for specimen selection.
RESUMO
BackgroundSurveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is essential for the global containment measures with regard to the ongoing pandemic. Diagnostic gold standard is currently reverse transcription of the (+)RNA genome and subgenomic RNAs and subsequent quantitative polymerase chain reaction (RT-qPCR) from nasopharyngeal swabs or bronchoalveolar lavages. In order to further improve the diagnostic accuracy, particularly for the reliable discrimination between negative and false-negative specimens, we propose the combination of the RT-qPCR workflow with subsequent pyrosequencing of a S-gene amplicon. This extension might add important value mainly in cases with low SARS-CoV-2 load, where RT-qPCR alone can deliver conflicting results.s ResultsWe successfully established a combined RT-qPCR and S-gene pyrosequencing method which can be optionally exploited after routine diagnostics or for epidemiologic studies. This allows a more reliable interpretation of conflicting RT-qPCR results in specimens with relatively low viral loads and close to the detection limits of qPCR (CT values >30). After laboratory implementation and characterization of a best practice protocol we tested the combined method in a large pediatric cohort from two German medical centers (n=769). Pyrosequencing after RT-qPCR enabled us to uncover 6 previously unrecognized cases of pediatric SARS-CoV-2 associated diseases, partially exhibiting unusual and heterogeneous presentation. Moreover, it is notable that in the course of RT-qPCR/pyrosequencing method establishment we did not observe any case of false-positive diagnosis when confirmed SARS-CoV-2-positive specimens were used from foregoing routine testing. ConclusionsThe proposed protocol allows a specific and sensitive detection of SARS-CoV-2 close to the detection limits of RT-qPCR. Combined RT-qPCR/pyrosequencing does not negatively affect preceding RT-qPCR pipeline in SARS-CoV-2 diagnostics and can be optionally applied in routine to inspect conflicting RT-qPCR results.
