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]
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.

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.

This protocol is a modified version of Nikki Freed and Olin Silanders protocol. To get information such as Primers, visit their protocol. 

Nikki Freed, Olin Silander 2020. nCoV-2019 sequencing protocol (RAPID barcoding, 1200bp amplicon).doi: 10.1093/biomethods/bpaa014


Our peer-reviewed paper is available here: https://www.mdpi.com/2076-2607/9/12/2598

[STEPS]
SECTION: Sample selection, RNA isolation
1. For whole genome sequencing using combined RT-PCR, samples with a Ct <20 are suitable. Higher Ct values ≤26 can be suitable as well, but we strongly recommend verification of the amplicon quality utilizing gelelectrophresis and/or microcapillary electrophoresis (Agilent) after combined RT-PCR.
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.
SECTION: Multiplex RT-PCR
2. For Primer dilution and setup, visit Nikki Freed and Olin Silanders protocol.
SECTION: Multiplex RT-PCR
2.1. 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)   10 µL 
Luna Enzyme Mix (20X)                                                 1 µL 
Primer Pool1 or Pool2                                                    1 µL 
Sample                                                                   up to  8 µL       (depending on Ct Value and concentration)                                                                                                                                                                                                                                                                                                                    
Nuclease free H2O                                         fill up to   20 µL          

    
SECTION: Multiplex RT-PCR
2.2. Set-up the following program on the thermal cycler:                                              

Step                                            Temperature     Time                    Cycles

Reverse Transcription              55 °C       30 min     1
Heat Activation                         95 °C       1 min     1
Denaturation                             95 °C        20 s     34 for Pool 1, 30 for Pool 2
Annealing and Extension        60 °C        210 s     34 for Pool 1, 30 for Pool 2
Final Extension                         65 °C        5 min 
Hold                                            4 °C            Indefinite             1


 

SECTION: Pooling and PCR quantification/normalisation
3. 
 

SECTION: Pooling and PCR quantification/normalisation
3.1. Quantify each PCR reaction using a Qubit or other method. Quantification using Nanodrop is not recommended.

 
  

SECTION: Pooling and PCR quantification/normalisation
3.2. Pool the two PCR reactions for each sample in a new PCR tube using 200 ng for each sample to a total amount of 400 ng . Add nuclease free water to a total volume of 7.5 µL .
SECTION: Rapid barocoding using the SQK RBK004
4. 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. 

 


SECTION: Rapid barocoding using the SQK RBK004
4.1. Set up the following reaction for each sample:


Component                                                                                                            Volume
DNA amplicons from step 15  (100ng total)                                                         7.5 µL
Fragmentation Mix RB01-12 (one for each sample, included in kit)                 2.5 µL
Total                                                                                                                             10 µL


  For higher number of samples it may be more efficient, to use SQK-RBK110.96 with 96 Barcodes, to come to a cost of  .

SECTION: Rapid barocoding using the SQK RBK004
4.2. Mix gently by flicking the tube, and spin down.



SECTION: Rapid barocoding using the SQK RBK004
4.3. Incubate the reaction in a PCR machine:

30 °C     for     1 min
80 °C     for      1 min
4 °C        for      30 s

SECTION: Rapid barocoding using the SQK RBK004
4.4. Pool all barcoded samples, noting the total volume.
SECTION: Rapid barocoding using the SQK RBK004
5. Ampure XP Bead Cleanup. Use a 1:1 ratio of sample to beads.

 

SECTION: Rapid barocoding using the SQK RBK004
6. Add 1 µLof RAP (from the RBK004 kit)  to 10 µLcleaned, barcoded DNA from previous step . Mix gently by flicking the tube, and spin down.

SECTION: Rapid barocoding using the SQK RBK004
7. Incubate the reaction for 5 min at room temperature.
SECTION: Rapid barocoding using the SQK RBK004
8. 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.
SECTION: MinION sequencing
9. 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. 

 

 
  


SECTION: MinION sequencing
10. 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.  

SECTION: MinION sequencing
11. 
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 (James Ferguson et Al). 

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'

Our sequences are available: https://bigd.big.ac.cn/gsa/browse/CRA005542

All data from our SEQs is available: https://t1p.de/seqdata