Abstract

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, as it is mobile, scalable, and cost-effective. Therefore, streamlined nanopore-sequencing protocols need to be developed and optimized for SARS-CoV-2 variants identification. 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. 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 the CT value as a useful pre-analytical parameter, which may help to decrease sequencing failures rates. Complete pipeline duration was approx. 7 h for one specimen and approx. 11 h for 12 multiplexed barcoded specimens. The adapted protocol contains fewer processing steps and can be completely conducted within one working day. Diagnostic CT values deduced from qPCR standardization experiments can act as principal criteria for specimen selection. As a guideline, SARS-CoV-2 genome copy numbers lower than 4 × 106 were associated with a coverage threshold below 20-fold and incompletely assembled SARS-CoV-2 genomes. 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.

Highlights

  • IntroductionTo face the ongoing SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2)

  • A particular focus is given to the identification of variants of concern (VOC) with accelerated transmission rates, increased infectivity or immune escape mutations as these variants would warrant adaptations in containment and vaccination strategies [1,2,3,4]

  • We tested whether specimens can be directly taken from be successfully primed by SARS-CoV-2-specific primers, which are subsequently used for residual diagnostic specimens extracted from 96-deepwell-plates using magnetic beads multiplex 1200 bp amplicon amplification

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Summary

Introduction

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). 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 [1,2,3,4]. The search for the zoonotic origin of SARS-CoV2 from comparative analyses of genomic data is an ongoing issue with relevance for the early recognition of future outbreak scenarios [5,6].

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