SubARTIC ONT SARS-CoV-2 Spike sequencing protocol (LoCost) V3.2

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.

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 10µL.

A	B
Component	Volume
LunaScript RT SuperMix (5X)	2 µL
Template RNA	8 µL

Incubate the reaction as follows:

25°C for 0h 2m 0s

55°C for 0h 10m 0s

95°C for 0h 1m 0s

Hold at 4°C

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 8µLof this master mix in to each well.

A	B	C
Component	Master mix O	Master Mix E
Working primer pool O	1.75 μl	-
Working primer pool E	-	1.75 μl
Q5® Hot Start High-Fidelity 2X Master Mix	6.25 μl	6.25 μl
Add to well	8μl	8 μl

Add 4.5µL 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 12.5µL.

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

Step Temperature Time Cycles

Heat Activation 98°C 0h 0m 30s 1

Denaturation 98°C 0h 0m 15s 35

Annealing 60°C 0h 5m 0s 35

Hold 4°C Indefinite 1

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

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

A	B
Reagent	Volume
Nuclease-free water	5 µl
NEB Ultra II End-prep reaction buffer	1.2 μl
NEB Ultra II End-prep enzyme mix	0.5 μl
Add to well	6.7 μl

Add 3.3µL 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.

Incubate the reaction using the following program (with a heated lid of 75 °C):

Step Temperature Time Cycles

Incubate 20°C 0h 20m 0s 1

Denaturation 65°C 0h 15m 0s 1

Hold 4°C Indefinite 1

Add 1.25µL 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.

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

A	B
Reagent	Volume
NEB Blunt/TA ligase Master Mix	5 μl
Nuclease-free water	2.75 μl
Add to well	7.75 μl

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

Incubate the reaction using the following program (with a heated lid at 75 °C):

Step Temperature Time Cycles

Incubate 20°C 0h 30m 0s 1

Denaturation 65°C 0h 10m 0s 1

Hold 4°C Indefinite 1

If you have 12-16 samples - then pool 10µL of each sample into a 1.5 ml DNA LoBind tube.

or

If you have 17-24 samples - then pool 7.5µLof each sample into a 1.5 ml DNA LoBind tube.

Resuspend the AMPure XP beads by vortexing. Add 1.0x volumes of resuspended AMPure XP beads to the reaction and mix by pipetting.

Incubate for Incubate for 0h 10m 0sat Room temperature

Prepare 1mLof fresh 80% ethanol with nuclease-free water.

Spin down the sample and pellet the beads on a magnet for . 0h 5m 0s. Keep the tube on the magnet, and pipette off the supernatant.

Take the sample off the magnet and add Short Fragment Buffer (SFB). 250µL 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.

Repeat the previous step ( 250µL wash with SFB , pellet, and then discard supernatant).

Keep the tube on the magnet and wash the beads with 250µL of freshly-prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

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.

Remove the tube from the magnetic rack and resuspend pellet in nuclease-free water. 30µL nuclease-free water. Gently pipette mix.

Incubate for 0h 2m 0s at Room temperature

Pellet the beads on a magnet until the eluate is clear and colourless. Remove and retain 28µLof eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

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 25µL.

A	B
Reagent	Volume
Clean Pooled barcoded sample	15 μl
Adapter Mix II (AMII)	2.5 μl
NEBNext Quick Ligation Reaction Buffer (5X)	5 μl
NEB Quick T4 DNA Ligase	2.5 μl

Spin down and incubate the reaction for 0h 20m 0sat Room temperature

Add 25µLof well mixed AMPure XP beads (1.0x) to the reaction and mix by pipetting. Incubate for 0h 10m 0s at Room temperature

Spin down the sample and pellet the beads on a magnet for 0h 5m 0s. Keep the tube on the magnet, and pipette off the supernatant.

Take the sample off the magnet and add Short Fragment Buffer (SFB). 125µL 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.

Repeat the previous step ( 125µL wash with SFB , pellet, and then discard supernatant).

Spin down and place the tube back on the magnet. Pipette off any residual SFB. Allow to air dry for ~0h 2m 0s, but do not dry the pellet to the point of cracking.

Remove the tube from the magnetic rack and resuspend pellet in Elution Buffer (EB). 15µLElution Buffer (EB).

Incubate for 0h 2m 0s at Room temperature

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

Quantify 1µLof 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.

Dilute if necessary to be ready to load ~15ng on the Flow Cell. 12µLof the final library will be loaded.

Add 30µLFLT to FB and mix well.

Rotate inlet port cover of the Flow Cell clockwise by 90 degrees so the inlet port is visible.

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.

Take 820µL 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.

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

Incubate for0h 5m 0s

Whilst incubating prepare the library as follows. Total volume in the tube will be 75µL.

A	B
Reagent	Volume
Final library (Max ~15ng total)	12 μl
SQB	37.5 μl
LB	25.5 μl

Gently lift the SpotON cover to open the SpotOn port.

Slowly load another 210µLof 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.

Pipette mix the library mixture together just prior to loading, as the loading beads can quickly settle.

Load the 75µLof mixture to the flow cell via the SpotON port in a dropwise fashion.

Gently replace the SpotOn port cover, making sure the bung sits in correctly, close the inlet port and close the GridION lid.

Select Start – Start sequencing.

Name the run and select the correct X position.

Choose the flowing options – LSK109 – NBD104 and NBD114.

Run for 16-24 hours, align to a Spike reference, select high-accuracy base calling, and set the minimum barcoding score to 80.

Start the run.