Sub-ARTIC Illumina SARS-CoV-2 Spike sequencing protocol (LoCost) V3.2

In a freshly bleached Pre-PCR hood (ideally in an isolated clean room), that has been subjected to UV irradiation for 0h 40m 0s, add LunaScript and RNA sample to a tube/well as follows (making sure to include negative controls):

A	B
Component	Vol/Rxn
LunaScript Super Mix	2 µl
RNA sample	8 µl

Mix thoroughly by pipetting up and down several times, seal plate, and centrifuge briefly.

Incubate the reaction as follows (with heated lid):

A	B
Temperature	Time
25°C	2 min
55°C	10 min
95°C	1 min
4°C	Hold

Primers are separated into two pools, odd and even, depending on where they sit across the Spike region. See the guidelines section for further details. In a clean pre-PCR hood set up two PCR reactions, one per pool as follows:

A	B	C
Component	Rxn 1	Rxn 2
Pool Even	0 µl	1.75 µl
Pool Odd	1.75 µl	0 µl
Q5 Hot Start Hi-Fi 2x Master Mix	6.25 µl	6.25 µl
Total	8 µl	8 µl

Add 4.5µL of cDNA to respective wells to give a total volume of 12.5µL. Include negative controls. Mix by pipetting, seal plate and centrifuge briefly.

Perform PCR using the following program (with heated lid):

A	B	C	D
Step	Temperature	Time	Cycles
Initial Denaturation	98°C	30 s	1
Denaturation	98°C	15 s	35
Annealing & Extension	60°C	5 min
Hold	4°C	Hold

In a freshly bleached post-PCR hood that has been subjected to UV for 0h 40m 0s, combine Pool Even and Pool Odd to give 20µL in each tube/well. Add 20µL nuclease-free water to dilute products 1 in 2.

Check the quality and concentration of negatives and a selection of samples using a fluorometer and/or Agilent TapeStation.

Prepare a master mix of the reagents as below by multiplying volumes by the number of samples; add 10% to allow for pipetting error.

A	B
Component	Vol/PCR Rxn
NEBNext Ultra II End Prep Enzyme Mix	0.6 µl
NEBNext Ultra II End Prep Reaction Buffer	1.4 µl
Total	2 µl

Mix well by pipetting and centrifuge briefly.

Combine 2µL of End Prep master mix with 10µL of amplified cDNA and mix by pipetting. Spin down, seal, place in a thermocycler and run the following program:

A	B
Temperature	Time
20°C	15 min
65°C	15 min
4°C	Hold

Prepare adapter ligation master mix by adding volumes as detailed below for each sample; adding 10% to allow for pipetting error.

A	B
Component	Vol/PCR rxn
NEBNext Ultra II Ligation Master Mix	6 µl
NEBNext Ligation Enhancer	0.2 µl
NEBNext Adapter	0.5 µl
Total	6.7 µl

Add 6.7µL ligation mix to each 12µL amplified cDNA/mastermix and mix by pipetting. Incubate at 20°C for 0h 15m 0s.

Add 1µL of USER enzyme to the ligation mixture. Mix by pipetting, centrifuge briefly and incubate at 37°C for 0h 15m 0s.

Increase volume of sample from 19.7µL to 25µL by the addition of nuclease-free water.

Perform a 0.9x Ampure XP bead clean by adding 22.5µL of Ampure XP beads and mix by pepetting. Incubate for 0h 5m 0s at 37Room temperature.

Transfer tube/plate to magnet and allow beads to clump for 0h 5m 0s. Remove supernatant taking care not to disturb pellet, and discard.

Wash the beads with 100µL 80% ethanol for 0h 0m 30s then remove ethanol by pipetting. Repeat one more time making sure to remove any residual ethanol.

Allow beads to air dry for around 0h 5m 0s; ensure that all the ethanol has evaporated. Do not let beads dry to the point of cracking, as this could affect the amount of DNA recovered.

Add 17µL of nuclease-free water to each well and mix by pipetting. Allow to stand for 0h 5m 0s then transfer to magnet and allow beads to clump for 0h 5m 0s. Remove 15µL into a fresh low-bind tube.

Perform Qubit assay to determine concentration.

Add the following components to separate wells of a PCR plate. Make sure to use the indexes in appropriate wells so that each sample has unique i5 and i7 indexes. If using less than 12 samples, the i5 primer can be replaced by the Universal PCR primer.

A	B
Component	Vol/Rxn
Adapter ligated cDNA fragments	7.5 µl
NEBNext Ultra II Q5 Master Mix	12.5 µl
Index Primer i7	2.5 µl
Index Primer i5*	2.5 µl
Total	25 µl

• if using 12 samples or fewer replace index primer i5 with the Universal primer.

Mix thoroughly by pipetting and centrifuge briefly.

Place on thermocycler and perform PCR using the following program:

A	B	C	D
Cycle Step	Temperature	Time	Cycles
Initial Denaturation	98°C	30 s	1
Denaturation	98°C	10 s	3 - 15*
Annealing/Extension	65°C	75 s
Final Extension	65°C	5 mins	1
Hold	4°C	Hold

*Refer to NEBNext protocol for number of cycles required. (Examples: 100 ng = ~3 cycles, 50 ng = ~3–4 cycles, 10 ng = ~6–7 cycles)

Pool 5µL of each sample together in a fresh low-binding microfuge tube and mix. This will give a final volume of 60µL for twelve samples. If larger numbers of samples are used then pool 5µL of each and mix. Split into aliquots of about 120µL.

Perform a 0.7x bead clean by adding 42µL or 84µL magnetic beads, depending on sample quantity. Leave for 0h 5m 0s to allow beads to bind DNA.

Place on a magnet and allow beads to clump for 0h 5m 0s. Remove supernatant and discard.

Wash pellet with 100µL 80% ethanol for 0h 0m 30s and remove. Repeat one more time. Allow the ethanol to dry off for about 0h 5m 0s, making sure the pelleted beads do not dry out, which can reduce DNA recovery.

Add 30µL of nuclease-free water and mix by pipetting. Leave for 0h 5m 0s at 37Room temperature to allow DNA to elute from the beads.

Place the tube/plate on the magnet and let the beads clump for 0h 5m 0s. Elute DNA into a fresh low-binding tube.

Perform TapeStation assay to determine if primer dimers are present. If present, perform another 0.7x bead clean and check again.

Perform qPCR by using a Kapa Library Quant kit for Illumina from Roche. Reactions are scaled down to 10µL. Use the appropriate Kapa kit for the type of qPCR machine used, see https://pim-eservices.roche.com/eLD/api/downloads/ca670ceb-fb38-eb11-0291-005056a71a5d?countryIsoCode=pi

Enter the values obtained from qPCR into the KAPA Library Quantification Data Analysis Template.

Dilute sample to 4nanomolar (nM).

Take 5µL of 4nanomolar (nM) dilution of library and add 5µL of 0.1Molarity (M) NaOH to denature the DNA and incubate at 37Room temperature for 0h 5m 0s.

Neutralise with 5µL of 200millimolar (mM) Tris-HCl 7.0 for 0h 1m 0s.

Add 985µL cold HT1 (hybridisation buffer) to give 20picomolar (pM) denatured library

Take35µL of 20picomolar (pM) sample and add 465µL of HT1. This will give 500µL of 1.4picomolar (pM)sequencing mix.

Thaw a tube of 10nanomolar (nM) PhiX stock .

Combine the following volumes in a microcentrifuge tube:

10nanomolar (nM) PhiX (10 μl) and RSB (Resuspension Buffer) (15 μl). The total volume is 25 μl at 4nanomolar (nM).

Vortex briefly and then pulse centrifuge. The 4nanomolar (nM) PhiX can be stored frozen for 3 months.

Combine 5µL of 4nanomolar (nM) PhiX with 5 μl of 0.1Molarity (M) NaOH. Vortex briefly and pulse centrifuge. Incubate at 37Room temperaturefor 0h 5m 0s

Add 5µL of 200nanomolar (nM) Tris-HCl (7.0), vortex briefly, pulse centrifuge and incubate at 37Room temperature for 0h 1m 0s

Add 985µLof HT1 (hybridisation buffer) to give a concentration of 20picomolar (pM). Vortex briefly and pulse centrifuge.

PhiX control at 20picomolar (pM) can be frozen at -20°C for up to two weeks. After this time cluster numbers tend to decrease.

Combine your library with PhiX control to give a final dilution of PhiX of 5%