ARTIC SARS-CoV-2 sequencing protocol v4 (LSK114)

Prepare between 11 and 95 RNA samples plus 1 negative control of nuclease-free water per library. If previously frozen, mix by briefly vortexing and pulse spin to collect liquid. Keep samples on ice at all times.

Mix the following components in a PCR strip-tubes/plate. Gently mix by pipetting and pulse spin the tube to collect liquid.

A	B
Component	Volume
LunaScript RT SuperMix (5X)	2 µL
Template RNA	8 µL
Total	10 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

If making up primer pools from individual oligos fully resuspend lyophilised oligos in 1xTE to a concentration of 100micromolar (µM), vortex thoroughly and spin down.

Sort all odd regions primers into one or more tube racks. Add 5µL of each odd region primer to a 1.5mLEppendorf tube labelled "Pool 1 (100micromolar (µM))". Repeat the process for all even region primers for Pool 2. These are your 100micromolar (µM) stocks of each primer pool.

Dilute 100micromolar (µM) pools 1:10 in molecular grade water, to generate 10micromolar (µM) primer stocks.

Set up the two PCR reactions per sample as follows in strip-tubes or plates. Gently mix by pipetting and pulse spin the tube to collect liquid at the bottom of the tube.

A	B	C
Component	Reaction 1	Reaction 2
5X Q5 Reaction Buffer	5 µL	5 µL
10 mM dNTPs	0.5 µL	0.5 µL
Q5 Hot Start DNA Polymerase	0.25 µL	0.25 µL
V3 Pool 1 (10µM)	4 µL	0 µL
V3 Pool 2 (10µM)	0 µL	4 µL
Nuclease-free water	12.75 µL	12.75 µL
Total	22.5 µL	22.5 µL

For M0493

or

A	B	C
Component	Reaction 1	Reaction 2
Q5 Hot Start High-Fidelity 2X Master Mix	12.5 µL	12.5 µL
V3 Pool 1 (10µM)	4 µL	0 µL
V3 Pool 2 (10µM)	0 µL	4 µL
Nuclease-free water	6 µL	6 µL
Total	22.5 µL	22.5 µL

For M0494

Add 2.5µL 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.

µL Set-up the following program on the thermal cycler:

Step Temperature Time Cycles

Heat Activation 98°C 0h 0m 30s 1

Denaturation 98°C 0h 0m 15s 25-35

Annealing 65°C 0h 5m 0s 25-35

Hold 4°C Indefinite 1

Optional step : If you wish you can check the DNA concentration of your reactions before proceeding, to determine successful amplification (or failure in the case of the negative control). If you are processing many samples, to save time, you can check the concentration of a few samples to check amplification success. In our experience, reactions are typically around 80-100ng/μL. If your samples are <50ng/μL you can double the amount used in the next step. We recommend quantifying DNA with a fluorometer, such as a Qubit or Quantus.

Label strip-tubes/plate and combine the following volumes of each PCR reaction for 10µL each sample:

A	B
Component	Volume
Pool 1 PCR reaction	2.5 µL
Pool 2 PCR reaction	2.5 µL
Nuclease-free water	45 µL
Total	50 µL

Barcode the amplicon pools using the one-pot native barcoding approach.

One-pot native barcoding of amplicons v4 (LoCost)

Quantify the barcoded amplicons using a fluorometer such as a Qubit or Quatus. Concentration will vary depending on number and Ct of samples and but you need about 30ng total at this stage to achieve maximum run yield.

Set up the Native Adapter (NA) ligation, using short fragment buffer (SFB) for clean-up, following the current ONT protocol for the kit you have purchased.

Quantify the barcoded amplicons using a fluorometer such as a Qubit or Quatus. Concentration will vary depending on number and Ct of samples but 15ng final library is usually required to acheive maximum run yield.

Prime and load the flowcell with 15ng of sequencing library, following the current ONT protocol for the kit you have purchased.

Start the sequencing run using MinKNOW.

Plug the MinION into the computer with the flowcell loaded, and wait for it to be detected.

Follow the sequential screens on MinKNOW to name the run, and select the appropriate kit(s). You should select ‘barcode both ends’, and we also recommend using the ‘trim barcodes’ function. If your computer has sufficient processing you can select ‘live basecalling’ (recommended), otherwise basecalling can be performed afterwards. We recommend using the High Accuracy basecalling mode.