TITLE: ARTIC SARS-CoV-2 sequencing protocol v4 (LSK114)
AUTHORS: Josh Quick, Lauren Lansdowne

[DESCRIPTION]
Amplicon sequencing protocol for SARS-CoV-2 v4 (LSK114)

We thank the ARTIC network, Oxford Nanopore Technologies, New England Biolabs, BCCDC, COG-UK, CanCOGen and protocols.io commenters for their assistance developing this protocol.

Changes in this version:

-Up to 95 samples per run with EXP-NBD196 native barcode kit
-Substitution of SuperScript IV for LunaScript RT SuperMix and reaction volume reduced to 10 uL.
-Substitution of Ultra II Ligation Module for Blunt/TA Ligase Master Mix and reaction volume reduced to 10 µL.
-Native barcode ligation reaction volume reduced to 10 uL.
-SFB wash volume reduced.

[BEFORE_START]
Prepare between 11 and 95 RNA samples plus 1 negative control using this protocol.

[STEPS]
SECTION: cDNA preparation
2. Mix the following components in a PCR strip-tubes/plate. Gently mix by pipetting and pulse spin the tube to collect liquid.

 

 

 

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
7. 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.


 

or

 

 

 

SECTION: Multiplex PCR
8. Add 2.5 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.
 

 

SECTION: Multiplex PCR
9.   Set-up the following program on the thermal cycler:  

Step                        Temperature     Time                    Cycles

Heat Activation     98 °C        30 s    1
Denaturation         98 °C        15 s     25-35 
Annealing              65 °C        5 min     25-35
Hold                       4 °C            Indefinite              1


 


 

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

 

 

 

SECTION: Native barocoding
11. Barcode the amplicon pools using the one-pot native barcoding approach.
 

SECTION: Native barocoding
12. 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 30 ng  total at this stage to achieve maximum run yield.


SECTION: Native barocoding
13. Set up the following Native Adapter (NA) ligation and clean-up with SFB.



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


 


SECTION: MinION sequencing
15. Prime the flowcell and load 15 ng sequencing library onto the flowcell.




 

SECTION: MinION sequencing
16. Start the sequencing run using MinKNOW.

 

 



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


SECTION: Primer pool preparation (optional)
6. Dilute 100 micromolar (µM)  pools 1:10 in molecular grade water, to generate 10 micromolar (µM) primer stocks.

 

 

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

 

 

SECTION: cDNA preparation
1. 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.
 

 

SECTION: Multiplex PCR
9.1. 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.