Protocol Implementation Considering Differences in Viral Loads and Influences of RNA Extraction Protocols Moreover, implementation of the interARTIC interface should help to avoid command line-based bioinformatics analyses as much as possible to provide a user-friendly and efficient analysis pipeline. We aimed at improving the efficiency of the nanopore sequencing workflow by using the onboard Guppy basecalling capability of the Oxford Nanopore MinION Mk1C device. Purified RNA from the different extraction protocols of milestone 1 was used for cDNA synthesis and successive multiplex PCR with the 2 ‘midnight’ split 1200 bp amplicon primer pools in single tube reactions. We tested whether reverse transcription can be successfully primed by SARS-CoV-2-specific primers, which are subsequently used for multiplex 1200 bp amplicon amplification. For comparison, we applied RNA purification protocols using silica columns (QiaAmp Viral Mini Kit, Qiagen) or guanidinium isothiocyanate (GITC) for RNA extraction (QIAzol lysis reagent, Qiagen). We tested whether specimens can be directly taken from residual diagnostic specimens extracted from 96-deepwell-plates using magnetic beads (Seegene NIMBUS/Tanbead). We defined milestones to simplify the protocol and decrease hands-on sample time and working step numbers. Library preparation was done using the Rapid Barcoding Sequencing Kit (SQK-RBK004 Oxford Nanopore Technologies, Oxford, UK) upon manufacturer’s recommendations.
Thereafter, amplicons from primer pools 1 and 2 were quantified using the QuantiFluor dsDNA System (Promega, Madison, WI, USA) with the Promega Quantus fluorometer and then mixed at equal concentrations. During the implementation phase, amplicon sizes and DNA concentrations were routinely checked by agarose gels or by microvolume electrophoresis using an Agilent Bioanalyzer instrument and a microfluidic chip (Agilent DNA 12,000 kit, Agilent). Then 34 cycles (pool 1) or 30 cycles (pool 2) of denaturation at 95 ☌ for 20 s and annealing and extension in one step at 60 ☌ for 210 s were performed. Reverse transcription was performed at 55 ☌ for 30 min, followed by incubation at 95 ☌ for one minute. 1 µL of 100 µM primer pool was used in each reaction. For RT-PCR, 8 µL of purified template RNA were used for each reaction.
#Oxford nanopore midnight protocol full
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.Ĭombined reverse transcription and amplification of multiple 1200 bp amplicons (RT-PCR) were performed in single tube 20 µL reactions using the Luna One-Step RT-qPCR Kit (NEB E3005). 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.
#Oxford nanopore midnight protocol portable
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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