TITLE: nanopore nCoV-2019 sequencing protocol (RAPID barcoding, 1200bp amplicon, combined RT-PCR)
AUTHORS: Anton  Pembaur, Erwan   Sallard, Patrick Weil, Jennifer Ortelt, Parviz Ahmad-Nejad, Jan Postberg

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">We established a protocol for fast, cost efficient Sars-CoV-2 sequencing with little as possible hands-on time (around 3h in total, excluding RNA extraction). The whole Sequencing can be done in one working day, including the bioinformatic pipeline. The cost per sample accumulates at around 40$, with already isolated RNA.</div><br/><div class = "text-block">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 all of the SARS-CoV-2 genome. Subsequently, we applied the Oxford Nanopore Rapid barcoding protocol and the portable MinION Mk1C sequencer in combination with the ARTIC bioinformatics pipeline. We tested the simplified and less time-consuming workflow on confirmed SARS-CoV-2-positive specimens from clinical routine and identified pre-analytical parameters, which may help to decrease the rate of sequencing failures. Duration of the complete pipeline was approx. 7 hrs for one specimen and approx. 11 hrs for 12 multiplexed barcoded specimens.</div><br/><div class = "text-block"><span>This protocol is a modified version of Nikki Freed and Olin Silanders </span><a href="http://dx.doi.org/10.17504/protocols.io.bgggjttw" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">protocol</span></a><span>. To get information such as Primers, visit their protocol. </span></div><br/><div class = "text-block">Nikki Freed, Olin Silander 2020. nCoV-2019 sequencing protocol (RAPID barcoding, 1200bp amplicon).doi: 10.1093/biomethods/bpaa014</div><br/></div>

[STEPS]
?. [Sample selection, RNA isolation]
For whole genome sequencing using combined RT-PCR, samples with a Ct Isolate RNA using any suitable protocol. We tried magnetic Bead based (Nimbus/TanBead), spin collum based (QIAamp Viral RNA, Qiagen) and trizol/chloroforme extraction, with no significant differences.
?. [Multiplex RT-PCR]
For Primer dilution and setup, visit Nikki Freed and Olin Silanders protocol.
?. [Multiplex RT-PCR]
Prepare RT-PCR reaction in a new PCR tube                                                                          Pipette Scheme for Luna Universal Probe One-Step RT-qPCR Kit (2X)  (Luna Probe One-Step RT-qPCR 4X Mix with UDG works too, adjust the volumes accordingly)Luna Universal Probe One-Step Reaction Mix (2X)   Luna Enzyme Mix (20X)                                                 Primer Pool1 or Pool2                                                    Sample                                                                   up to         (depending on Ct Value and concentration)                                                                                                                                                                                                                                                                                                                    Nuclease free H2O                                         fill up to
10 µl
1 µl
1 µl
8 µl
20 µl
?. [Multiplex RT-PCR]
Set-up the following program on the thermal cycler:                                              Step                                            Temperature     Time                    CyclesReverse Transcription                   1Heat Activation                              1Denaturation                                  34 for Pool 1, 30 for Pool 2Annealing and Extension             34 for Pool 1, 30 for Pool 2Final Extension                         Hold                                                        Indefinite             1
55 °C
95 °C
95 °C
60 °C
65 °C
4 °C
Final concentrations of PCR products can range from ~20-150ng/µl. We dont see unexpected products while using more than sufficient input material.
?. [Pooling and PCR quantification/normalisation]
At this stage, care should be taken with amplified PCR products. Only open tubes in a designated post-PCR workspace with equipment that is separate from areas where primers and mastermixes are handled. After combining the two pools of amplified DNA, the PCR products can be used for Oxford Nanopore Sequencing, using the RAPID barcode kit RBK004, as described in this protocol.Alternatively, these amplicons can be used for Oxford Nanopore Sequencing, following Josh Quick's ligation based protocol (CoV-2019 sequencing protocol v2, dx.doi.org/10.17504/protocols.io.bdp7i5rn, at step 15)  using the SQK-LSK109 kit.Alternatively, these amplicons can also be used for Illumina sequencing, such as found here: x.doi.org/10.17504/protocols.io.betejeje Silander et al stated that performing an Ampure XP bead clean up at this stage does not improve performance. Therefore, it is not necessary to clean up the PCR reaction at this step.
?. [Pooling and PCR quantification/normalisation]
Quantify each PCR reaction using a Qubit or other method. Quantification using Nanodrop is not recommended.
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?. [Pooling and PCR quantification/normalisation]
Pool the two PCR reactions for each sample in a new PCR tube using 200 ng for each sample to a total amount of  . Add nuclease free water to a total volume of  .
400 ng
7.5 µl
?. [Rapid barocoding using the SQK RBK004]
Multiple samples can be run on the same flow cell by barcoding. Up to 12 samples can be multiplexed in this approach. Amplicons from each sample will be individually barcoded in the following steps. These follow the RBK004 protocol from Oxford Nanopore.   Tip: aliquot the Rapid barcodes into a PCR strip to enable multichannelling.
?. [Rapid barocoding using the SQK RBK004]
Set up the following reaction for each sample:Component                                                                                                            VolumeDNA amplicons from step 15  (100ng total)                                                         Fragmentation Mix RB01-12 (one for each sample, included in kit)                 Total                                                                                                                              For higher number of samples it may be more efficient, to use SQK-RBK110.96 with 96 Barcodes, to come to a cost of .
7.5 µl
2.5 µl
10 µl
?. [Rapid barocoding using the SQK RBK004]
Mix gently by flicking the tube, and spin down.
?. [Rapid barocoding using the SQK RBK004]
Incubate the reaction in a PCR machine:     for          for              for
30 °C
80 °C
4 °C
?. [Rapid barocoding using the SQK RBK004]
Pool all barcoded samples, noting the total volume.
?. [Rapid barocoding using the SQK RBK004]
Ampure XP Bead Cleanup. Use a 1:1 ratio of sample to beads.
?. [Rapid barocoding using the SQK RBK004]
Add of RAP (from the RBK004 kit)  to cleaned, barcoded DNA from previous step . Mix gently by flicking the tube, and spin down.
1 µl
10 µl
?. [Rapid barocoding using the SQK RBK004]
Incubate the reaction for  at room temperature.
?. [Rapid barocoding using the SQK RBK004]
The prepared library is used for loading into the MinION flow cell according to Oxford Nanopore Rapid Barcoding (RBK004) protocol. Please refer to the Oxford Nanopore Rapid Barcoding RBK004 protocol at this stage. Store the library on ice until ready to load.
?. [MinION sequencing]
Start the sequencing run using MinKNOW. For real-time surveillance of basecalling and demultiplexing for each of the 12 multiplexed samples per run, enable these options in the MinKNOW software or use RAMPART (https://github.com/artic-network/rampart) on a dedicated LINUX environment.
?. [MinION sequencing]
Depending on the variation in coverage of each amplicon, generally, you will need approx 10,000 to 20,000 reads or 10-20Mb per sample to confidently assemble and call variants. This can typically be achieved on a minION flow cell in around four hours when runnning 12 samples. Shorter, if running fewer samples.
?. [MinION sequencing]
For a simple bioinformatic pipeline optimized on Sars-CoV-2 genome assembly and analysis, we recommend using the interARTIC tool, wich provides a GUI to ether medaka or nanopolish workflow. If needed, for more accurate variant calling, we wrote a python script which combines the consensus sequence from medaka and nanopolish pipeline. After downloading this tool  , direct to the save path of this tool in the command line of the console via cd command. Follow the example below, which, after python command, uses the arguments 'medaka_consesensus_sequence_path', 'nanopolish_consesensus_sequence_path', 'reference_genome_path' and 'output_path':python consensus_merging.py '/home/documents/interartic_medaka/nCoV_2019_midnight_V1_sample1_medaka_single_medaka/medaka.consensus.fasta' '/home/documents/interartic_nanopolish/nCoV_2019_midnight_V1_sample1_nanopolish_single_nanopolish/param2.consensus.fasta' '/home/documents/sarscov2reference.fasta' '/home/documents/merge_output'All data from our SEQs is available: https://t1p.de/seqdata