TITLE: SubARTIC ONT SARS-CoV-2 Spike sequencing protocol (LoCost) V3.2 
AUTHORS: Paul J Parsons, Gavin  Horsburgh, Kathryn        Maher, Steve      Paterson, Terry Burke

[DESCRIPTION]
This protocol describes a procedure for sequencing the Spike gene of SARS-CoV-2 using short amplicons (146-208bp) with Oxford Nanopore technology (R9.4.1 MinION/GridION Flow Cell). The method has proved to be successful with both clinical RNA samples and degraded wastewater samples. The primers are unique to the SubARTIC method. The library prep procedure has been heavily adapted from the ncov-2019 sequencing v3 (ARTIC) protocol by Josh Quick (https://www.protocols.io/view/ncov-2019-sequencing-protocol-v3-locost-bh42j8ye) and the "low cost" method from the NEOF Liverpool Illumina ARTIC protocol. See this link for the version of the protocol using Illumina sequencing (https://www.protocols.io/view/sub-artic-illumina-sars-cov-2-spike-sequencing-pro-btpjnmkn). 

[BEFORE_START]
  

Before starting, generate the "Odd" and "Even" primer pools as follows:


1. Fully resuspend lyophilised oligonucleotides in 1x TE to a concentration of 100 micromolar (µM) , vortex thoroughly and spin down.
2. Sort the odd and even primer sets into separate batches and label two 1.5-ml tubes.
3. Starting with the even primer set add the volume (μl) of stock primer given in the attached (above) to the pooled 1.5-ml tube (between 7.5μl - 15μl). Vortex and spin down. Then repeat the pooling with the odd primers. These are your two pooled stocks. 
4. Dilute the pool stocks one in ten across several aliquots with ultrapure water. Vortex and spin down. These are your working primer pools used in step 4. 
 


[GUIDELINES]
This protocol was devised using a R9.4.1 Flow Cell on a GridION, please be aware of any potential amendments needed for other sequencers/flow cell types. Consult the Nanopore community if you are in need of assistance https://nanoporetech.com/community. The primers are unique to this method. See   and the BEFORE STARTING section for details. 


[STEPS]
SECTION: cDNA preparation
1. Prepare between 11 and 23 RNA samples plus a negative control (nuclease-free water) per library. If previously frozen, mix by briefly flicking and pulse spin to collect liquid. Keep samples on ice at all times.

 

 

SECTION: cDNA preparation
2. Mix the following components in PCR strip-tubes/plate. Gently mix by pipetting and pulse spin the tube to collect liquid. Total volume per well is 10 µL.

 

 

SECTION: cDNA preparation
3. Incubate the reaction as follows:

 25 °C for 2 min
 55 °C for 10 min
 95 °C for 1 min
Hold at 4 °C 
SECTION: Multiplex PCR
5. Add 4.5 µL cDNA to each of the PCR reactions, gently mix by pipetting and pulse spin the tube to collect liquid at the bottom of the tube. Total volume per well is 12.5 µL. 


SECTION: Multiplex PCR
6. Run the following program on the thermal cycler; same PCR profile is used for both pools:  

Step                        Temperature     Time                    Cycles

Heat Activation     98 °C        30 s    1
Denaturation         98 °C        15 s     35 
Annealing              60 °C        5 min     35
Hold                       4 °C            Indefinite              1


 


SECTION: Pooling of O and E PCR products
7. Move to a cleaned and UV sterilised Post-PCR hood. Combines pool O and E of each sample. Dilute ~1 in 2 by adding 20 µL  nuclease-free water and pipette mix.

 

SECTION: Multiplex PCR
4. Primers are separated into two pools, Odd (O) and Even (E), depending on where they sit across the Spike region. Each sample is then amplified with these two pools separately and pooled after PCR. See the BEFORE STARTING section (above) for further details. Set up two master mixes (O and E) to cover your sample number with an excess of around 10%. Do not over-vortex the Q5. Dispense 8 µLof this master mix in to each well. 
 
 

SECTION: End prep
8. Create a master mix as listed below - one per pooled sample - make an excess of ~10%. Dispense 6.7 µL of this master mix to each well. 


 

SECTION: End prep
9. Add 3.3 µL of each pooled and diluted product (from step 7) to the end-prep reactions, gently mix by pipetting and pulse spin the tube to collect liquid at the bottom of the tube. 
SECTION: End prep
10. Incubate the reaction using the following program (with a heated lid of 75 °C):

Step                        Temperature     Time                    Cycles

Incubate                 20 °C        20 min    1
Denaturation         65 °C        15 min     1 
Hold                       4 °C            Indefinite              1


 

SECTION: Native Barcoding
11. Add 1.25 µL of each barcode to a new PCR strip/plate. One barcode per sample. Place the droplet on the side of the well to confirm it is present and then spin down.


SECTION: Native Barcoding
12. Create a master mix as listed below one per pooled sample. Make an excess of ~10%. Dispense 7.75 µL of this master mix to each well. 


 

 

SECTION: Native Barcoding
13. Once the end prep is complete, Add 1 µL of the end-prepped PCR product to each well. Mix by flicking and spin down. The total in each well will be 10 µL

SECTION: Native Barcoding
14. Incubate the reaction using the following program (with a heated lid at 75 °C):

Step                        Temperature     Time                    Cycles

Incubate                 20 °C        30 min    1
Denaturation         65 °C        10 min     1 
Hold                       4 °C            Indefinite              1
 

SECTION: One-pot Ampure bead clean 1
15. If you have 12-16 samples - then pool 10 µL of each sample into a 1.5 ml DNA LoBind tube.
or
If you have 17-24 samples - then pool 7.5 µLof each sample into a 1.5 ml DNA LoBind tube.
SECTION: One-pot Ampure bead clean 1
16. Resuspend the AMPure XP beads by vortexing. Add 1.0x volumes of resuspended AMPure XP beads to the reaction and mix by pipetting.

SECTION: One-pot Ampure bead clean 1
17. Incubate for Incubate for 10 minat Room temperature

 

SECTION: One-pot Ampure bead clean 1
18. Prepare 1 mLof fresh 80% ethanol with nuclease-free water.


SECTION: One-pot Ampure bead clean 1
19. Spin down the sample and pellet the beads on a magnet for 5 min. Keep the tube on the magnet, and pipette off the supernatant.

SECTION: One-pot Ampure bead clean 1
20. Take the sample off the magnet and add 250 µL Short Fragment Buffer (SFB). Resuspend beads completely by pipette mixing. Return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard.
SECTION: One-pot Ampure bead clean 1
21. Repeat the previous step (250 µL wash with SFB, pellet, and then discard supernatant).

SECTION: One-pot Ampure bead clean 1
22. Keep the tube on the magnet and wash the beads with 250 µL of freshly-prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

 

SECTION: One-pot Ampure bead clean 1
23. Spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to air dry for ~2 min, but do not dry the pellet to the point of cracking.
SECTION: One-pot Ampure bead clean 1
24. Remove the tube from the magnetic rack and resuspend pellet in 30 µL nuclease-free water. Gently pipette mix. 
 


SECTION: One-pot Ampure bead clean 1
25. Incubate for 2 min at Room temperature

SECTION: One-pot Ampure bead clean 1
26. Pellet the beads on a magnet until the eluate is clear and colourless. Remove and retain 28 µLof eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
 


SECTION: Adapter ligation
27. By adding directly to the pooled and barcoded sample, perform adapter ligation as follows. Make sure to mix by flicking the tube between each sequential addition. Total volume in the tube will be 25 µL.

 

SECTION: Adapter ligation
28. Spin down and incubate the reaction for 20 minat Room temperature

 

SECTION: Final library bead clean
29. Add 25 µLof well mixed AMPure XP beads (1.0x) to the reaction and mix by pipetting. Incubate for 10 min at Room temperature

 

SECTION: Final library bead clean
30. Spin down the sample and pellet the beads on a magnet for 5 min. Keep the tube on the magnet, and pipette off the supernatant.

SECTION: Final library bead clean
31. Take the sample off the magnet and add 125 µL Short Fragment Buffer (SFB). Resuspend beads completely by pipette mixing. Return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard.
SECTION: Final library bead clean
32. Repeat the previous step (125 µL wash with SFB, pellet, and then discard supernatant).
SECTION: Final library bead clean
33. Spin down and place the tube back on the magnet. Pipette off any residual SFB. Allow to air dry for ~2 min, but do not dry the pellet to the point of cracking.


 

SECTION: Final library bead clean
34. Remove the tube from the magnetic rack and resuspend pellet in 15 µLElution Buffer (EB).
 


SECTION: Final library bead clean
35. Incubate for 2 min at Room temperature 

SECTION: Final library bead clean
36. Pellet the beads on a magnet until the eluate is clear and colourless. Remove and retain 14 µL of eluate into a new clean 1.5 ml DNA LoBind tube.

SECTION: Final library bead clean
37. Quantify 1 µLof eluted sample using a Qubit fluorometer High sensitivity kit. – The sample is expected ~ 1-3 ng/ul range depending on the number of samples used.

SECTION: Final library bead clean
38. Dilute if necessary to be ready to load ~15ng on the Flow Cell. 12 µLof the final library will be loaded.
 


SECTION: Gridion Sequencing
39. Add 30 µLFLT to FB and mix well.
 


SECTION: Gridion Sequencing
40. Rotate inlet port cover of the Flow Cell clockwise by 90 degrees so the inlet port is visible.
SECTION: Gridion Sequencing
41. Take a P1000 and set the volume to 800 μl. Place the tip in the inlet port, make sure the pipette is held perpendicularly (i.e. not at an angle). Remove any air by turning the dial on the pipette slowly. You should see a small amount of liquid on the end of the tip. Do not remove more than necessary.
SECTION: Gridion Sequencing
42. Take 820 µL from the FLT/FB mix tube being careful that there are no air bubbles present/liquid goes all the way to the bottom of the tip. 

SECTION: Gridion Sequencing
43. Load this into the priming port by dispensing slowly. Save the last few μl in the pipette tip to avoid adding any air. 
 

SECTION: Gridion Sequencing
44. Incubate for5 min

SECTION: Gridion Sequencing
45. Whilst incubating prepare the library as follows. Total volume in the tube will be 75 µL.
 

 

SECTION: Gridion Sequencing
46. Gently lift the SpotON cover to open the SpotOn port.
SECTION: Gridion Sequencing
47. Slowly load another 210 µLof the FB/FLT mix into the Inlet port. This should initiate a siphon at the SpotON port. As previously, leave the last amount of liquid in the end of the tip to avoid any bubbles. 

SECTION: Gridion Sequencing
48. Pipette mix the library mixture together just prior to loading, as the loading beads can quickly settle. 
SECTION: Gridion Sequencing
49. Load the 75 µLof mixture to the flow cell via the SpotON port in a dropwise fashion. 

SECTION: Gridion Sequencing
50. Gently replace the SpotOn port cover, making sure the bung sits in correctly, close the inlet port and close the GridION lid.
SECTION: Starting the experiment
51. Select Start – Start sequencing.
SECTION: Starting the experiment
52. Name the run and select the correct X position. 
SECTION: Starting the experiment
53. Choose the flowing options – LSK109 – NBD104 and NBD114.
SECTION: Starting the experiment
54. Run for 16-24 hours, align to a Spike reference, select high-accuracy base calling, and set the minimum barcoding score to 80. 
 

SECTION: Starting the experiment
55. Start the run.