TITLE: nCoV-2019 sequencing protocol (single sample)
AUTHORS: Josh Quick

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">ARTIC amplicon sequencing protocol for MinION for nCoV-2019</div></div>

[STEPS]
?. [cDNA preparation]
Mix the following components in an 0.2mL 8-strip tube;Component                                        Volume50µM random hexamers                 10mM dNTPs mix (10mM each)    Template RNA                                   Total
1 µl
1 µl
11 µl
13 µl
Viral RNA input from a clinical sample should be between Ct 18-35. If Ct is between 12-15, then dilute the sample 100-fold in water, if between 15-18 then dilute 10-fold in water. This will reduce the likelihood of PCR-inhibition.
A mastermix should be made up in the mastermix cabinet and aliquoted into PCR strip tubes. Tubes should be wiped down when entering and leaving the mastermix cabinet.
?. [cDNA preparation]
Gently mix by pipetting and pulse spin the tube to collect liquid at the bottom of the tube.
?. [cDNA preparation]
Incubate the reaction as follows: for Place on ice for
65 °C
?. [cDNA preparation]
Add the following to the annealed template RNA: Component	                                Volume SSIV Buffer	                                 100mM DTT                                  RNaseOUT RNase Inhibitor 	 SSIV Reverse Transcriptase	 Total
4 µl
1 µl
1 µl
1 µl
20 µl
A mastermix should be made up in the mastermix cabinet and added to the denatured RNA in the extraction and sample addition cabinet. Tubes should be wiped down when entering and leaving the mastermix cabinet.
?. [cDNA preparation]
Gently mix by pipetting and pulse spin the tube to collect liquid at the bottom of the tube.
?. [cDNA preparation]
Incubate the reaction as follows:Hold at
42 °C
70 °C
5 °C
?. If required resuspend lyophilised primers at a concentration of 100µM each
nCov-2019/V2 primers for this protocol were designed using Primal Scheme and generate overlapping 400nt amplicons. Primer names and pools are listed in the .TSV file.
?. [Primer pool preparation]
Generate primer pool stocks by adding  of each primer pair to a  Eppendorf labelled either “Pool 1 (100µM)” or “Pool 2 (100µM)”. Total volume should be  for Pool 1 (100µM) and  for Pool 2 (100µM). These are your 100µM stocks of each primer pool.
5 µl
1.5 ml
490 µl
490 µl
Primers should be diluted and pooled in the mastermix cabinet which should be cleaned with decontamination wipes and UV sterilised before and after use.
?. [Primer pool preparation]
Dilute this primer pool 1:10 in molecular grade water, to generate 10µM primer stocks. It is recommend that multiple aliquots of each primer pool are made to in case of degradation or contamination.
Primers need to be used at a final concentration of 0.015µM per primer. In this case both pools have 98 primers in so the requirement is 3.6µL primer pools (10uM) per 25µL reaction. For other schemes, adjust the volume added appropriately.
?. [Multiplex PCR]
In the mastermix hood set up the multiplex PCR reactions as follows in 0.2mL 8-strip PCR tubes:Component                                             Pool 1                Pool 2 5X Q5 Reaction Buffer                           10 mM dNTPs                                         Q5 Hot Start DNA Polymerase             Primer Pool 1 or 2 (10µM)                    Nuclease-free water                              Total
5 µl
5 µl
0.5 µl
0.5 µl
0.25 µl
0.25 µl
3.6 µl
3.6 µl
13.15 µl
13.15 µl
22.5 µl
22.5 µl
A PCR mastermix for each pool should be made up in the mastermix cabinet and aliquoted into PCR strip tubes. Tubes should be wiped down when entering and leaving the mastermix cabinet.
?. [Multiplex PCR]
In the extraction and sample addition cabinet add  cDNA to each tube and mix well by pipetting.
2.5 µl
The extraction and sample addition cabinet should should be cleaned with decontamination wipes and UV sterilised before and after use.
?. [Multiplex PCR]
Pulse centrifuge the tubes to collect the contents at the bottom of the tube.
?. [Multiplex PCR]
Set-up the following program on the thermal cycler:  Step                        Temperature     Time                    CyclesHeat Activation         1Denaturation              25-35 Annealing                   25-35Hold                                   Indefinite              1
98 °C
98 °C
65 °C
4 °C
Cycle number should be 25 for Ct 18-21 up to a maximum of 35 cycles for Ct 35
?. [PCR clean-up]
Combine the entire contents of “Pool 1” and “Pool 2” PCR reactions for each biological sample into to a single Eppendorf tube.
1.5 ml
?. [PCR clean-up]
Clean-up the amplicons using the following protocol:
Amplicon clean-up should be performed in the post-PCR cabinet which should should be cleaned with decontamination wipes and UV sterilised before and after use.
?. [Quantification and normalisation]
Quantify the amplicon pools using the Quantus Fluorometer using the ONE dsDNA assay.
If the concentration is greater than 25 ng/µL dilute the sample by a factor of 10 by adding 270µL 10mM Tris and quantify again using the Quantus fluorometer.
?. [Quantification and normalisation]
Label a  Eppendorf tube for each sample.
1.5 ml
This is a ‘one-pot ligation’ protocol for native barcoded ligation libraries. We have seen no reduction in performance compared to standard libraries, and is made faster by using the Ultra II® ligation module which is compatible with the Ultra II® end repair/dA-tailing module removing a clean-up step.
?. [Quantification and normalisation]
Normalise the input by diluting each sample to . Use  input for the end-preparation reaction to give a total input of
10 µl
50 ng
Input to the one-pot native barcoding reaction will vary depending on the amplicon length but we have determined 5ng is the correct input for efficient barcoding of this amplicon length. Process at least 7 samples plus one negative control per native barcoded library in order to have sufficient material at the end.
?. [Library quantification]
Quantify the final library using the Quantus Fluorometer using the ONE dsDNA assay.
Final library can be now be stored in 10 mM Tris pH 8 at 4°C for up to a week if needed otherwise proceed directly to MinION sequencing.
?. [MinION sequencing]
Prime the flowcell and load  sequencing library onto the flowcell.
20 ng
From experience we know 20 ng is optimum loading input for short amplicons.
?. [MinION sequencing]
Start the sequencing run using MinKNOW.
?. [End-preparation]
Perform end-preparation on the single amplicon pool using the Ultra II End Repair/dA-Tailing module
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?. [End-preparation clean-up]
Clean-up end-preparation reaction using a 1x volume of SPRI beads and elute in  Elution Buffer (EB)
30 µl
?. [AMX ligation]
Perfom adapter ligation on the single amplicon pool with AMX.
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