TITLE: SARS-CoV-2 Amplicon-based Illumina Sequencing Protocol
AUTHORS: wgscov  

[DESCRIPTION]
ARTIC amplicon sequencing protocol adapted from Josh Quick's https://www.protocols.io/view/ncov-2019-sequencing-protocol-v2-bdp7i5rn for Illumina sequencing of SARS-CoV-2

[STEPS]
SECTION: Preamble to cDNA Synthesis
1. Use either a SuperScript (step 2) or LunaScript kit (step 3) for cDNA synthesis
SECTION: cDNA Synthesis
2. Option 1:  SuperScipt mastermix
SECTION: cDNA Synthesis
2.1. In a clean room, mix 1:1 dNTP and random hexamers. Aliquot 2 µL  per reaction, and add 11 µL  RNA, as per the table below. Seal plate and gently mix and centrifuge briefly to collect liquid at the bottom of the well.

Component	                                        Volume
50 µM random hexamers	               1 µL
10mM dNTPs mix (10mM each)      1 µL 
Total Mastermix volume                    2 µL
(template RNA)                                   11 µL
Total Reaction volume                      13 µL



The Mastermix should be prepared in a clean room and added to the nucleic acid in a BSC exclusive for RNA work.
SECTION: cDNA Synthesis
2.2. Incubate the reaction in a thermocycler as follows:

Step                        Temperature             Time                Cycles
1                             65 °C        5 min         1
2                             4 °C         Hold

SECTION: cDNA Synthesis
2.3. Prepare the following SuperScipt mastermix:


Component	                                        Volume
SSIV Buffer	                                       4 µL  
100mM DTT                                         1 µL
RNaseOUT RNase Inhibitor               1 µL
SSIV Reverse Transcriptase              1 µL
Total Mastermix volume                    7 µL
(denatured RNA)                                 13 µL
Total Reaction volume                       20 µL


Add 7 µL  of mastermix to the denatured RNA from the previous step. Cover the plate with seal, mix gently on a  plate mixer, and pulse spin the plate to collect liquid at the bottom of the tube.  


The Mastermix should be prepared in in a clean room and added to the denatured RNA in a BSC or workbench exclusive for RNA work.
SECTION: cDNA Synthesis
2.4. Incubate in a thermocycler as follows:

Step        Temperature            Time                    Cycles

1              42 °C         50 min         1
2              70 °C          10 min         1
3               4 °C          Hold

SECTION: cDNA Synthesis
3. Option 2: LunaScript mastermix
SECTION: cDNA Synthesis
3.1. In a clean room, mix the components as per the table below:

Component	                                        Volume
Nuclease-free water    	               5 µL
LunaScript RT SuperMix (5x)            4 µL 
Total Mastermix volume                    9 µL
(template RNA)                                   11 µL
Total Reaction volume                       20 µL

The Mastermix should be prepared in a clean room and added to the nucleic acid in a BSC exclusive for RNA work.
SECTION: cDNA Synthesis
3.2. Incubate the reaction in a thermocycler as follows:

Step                   Temperature             Time                    Cycles
1                       25 °C               2 min           1
2                       55 °C               20 min           1
3                        95 °C              1 min           1
4                       4 °C                  2 min          1

SECTION: Multiplex PCR Amplification
4. Prepare two multiplex PCR mastermixes as follows (1 for each pool):


Component                                                Pool 1                      Pool 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
ARTIC Primer Pool 1 or 2 (10µM)       3.6 µL              3.6 µL
Nuclease-free water                             13.15 µL         13.15 µL
Total  Mastermix volume                     22.5 µL           22.5 µL
(cDNA)                                                    2.5 µL             2.5 µL
Total reaction volume                          25 µL               2.5 µL

Prealiquot 22.5 µL   of each mastermix (pool1 and pool2) to each plate (pool1 and pool2) accordingly. Add 2.5 µL cDNA to each well of both plates. Seal the plate, mix gently and centrifuge briefly to collect liquid at the bottom of the well.


SECTION: Multiplex PCR Amplification
5. Run the 3.5 hours PCR program for each pool:

Step                        Temperature             Time                        Cycles
Heat Activation     98 °C            30 s                1
Denaturation          98 °C           15 s                 35 
Annealing             65 °C             5 min                 35
Hold                        4 °C                                                          1

SECTION: Amplicon Pooling and Cleanup
6. Pool 12.5 µL of each pool 1 and 2 together (total 25μl) in an 0.2 ml 96 well PCR plate.

Perform AMPure XP bead cleanup according to directions, as follows.
SECTION: Amplicon Pooling and Cleanup
6.1. Add 25 µL of AMPure XP (well-vortexed, room temperature) to the sample plate. Cover the plate with seal, mix gently on a  plate mixer, and pulse spin the plate to collect liquid at the bottom of the tube. Incubate at Room temperature  for 5 min.

SECTION: Amplicon Pooling and Cleanup
6.2. Place the plate on a magnetic rack for 5 min , or until the beads have pelleted and the supernatant is completely clear.

SECTION: Amplicon Pooling and Cleanup
6.3. Remove and discard the liquid from each well with a multichannel pippette, being careful not to touch the bead pellet. 
SECTION: Amplicon Pooling and Cleanup
6.4. Add 200 µL  of freshly prepared, room temperature  80% ethanol to each well, incubate for 30 s , remove the ethanol carefully with a multichannel pipette.

SECTION: Amplicon Pooling and Cleanup
6.5. Repeat ethanol wash (step 6.4).Discard all ethanol and carefully remove as much residual ethanol as possible using a multichannel pipette. With the plate uncovered, incubate for 3 min to 5 min or until the pellet loses its shine (if the pellet dries completely it will crack and become difficult to resuspend). 

SECTION: Amplicon Pooling and Cleanup
6.6. Remove from magnetic rack, add28 µL  of EB buffer to wells and mix gently on a plate mixer, ensuring beads are well re-suspended. Briefly centrifuge the plate to collect the liquid at the bottom of the wells. Incubate at room temperature   for 5 min .

SECTION: Amplicon Pooling and Cleanup
6.7. Place the plate on magnetic rack and incubate for 2 min  to 5 minor until the beads have pelleted and the supernatant is completely clear. 

SECTION: Amplicon Pooling and Cleanup
6.8. Transfer 25 µL  of the clear supernatant to a new plate, ensuring no beads are transferred. 

SECTION: Gel Electrophoresis
7. Optional section; use remaining volumes from Pool 1 and Pool 2 to confirm amplification by gel electrophoresis. 
SECTION: Gel Electrophoresis
7.1. Prepare 1% agarose gels with enough wells to load all samples. Load 2 µLof a 100 bp ladder into gel on either side of each row of wells.

SECTION: Gel Electrophoresis
7.2. Dispense 2 µL   of 6X loading dye into each sample with a multichannel pipette, mix and load 2 µL   of this mix into the gel. 

SECTION: Gel Electrophoresis
7.3. Run at 240V for 20 min. Visualize PCR products, confirm bands of approximately 300bp size.

SECTION: Amplicon Quantification and Normalization
8. Quantify amplicons using Qubit dsDNA High Sensitivity kit and plate reader according to directions, as follows.
SECTION: Amplicon Quantification and Normalization
8.1. Create Qubit dsDNA HS working solution by mixing 99.5 µL X  buffer and 0.5 µLX dye (X is the total number of samples, including 6 standards). Using a reservoir and multichannel pipette, dispense  98 µL   into required number of wells of a Costar 3590 flat-bottom plate (or as appropriate for plate reader).

SECTION: Amplicon Quantification and Normalization
8.2. Dilute the clean, pooled amplicons (from step 6.8) 1:10 by mixing3 µL  of the amplicons in 27 µL  of nuclease free water.

SECTION: Amplicon Quantification and Normalization
8.3. Make up serial standards using 1:2 dilutions of 10 ng/ul stock (Standard 2) from the Qubit HS. This creates 5 standards in the following concentrations: 10 ng/ul  5 ng/ul 2.5 ng/ul 1.25 ng/ul 0.625 ng/ulplus Standard 1 0 ng/ul .

SECTION: Amplicon Quantification and Normalization
8.4. Mix2 µLof diluted amplicons and each of the 6 standards, 98 µL  of Qubit HS working solution, mix and breifly centrifuge. Use plate reader to obtain concentration reading for each sample and standards. The Qubit standard curve is generated by the Qubit standards.

SECTION: Amplicon Quantification and Normalization
9. Based on the amplicon concentration, normalize of all the samples amplicon concentration to 0.2 ng/ul.
This can be done by adding 2.5 µL  of diluted amplicon to a plate with prealiquoted, appropriate amount of nuclease free water. 

SECTION: Library Preparation
10. Prepare sequencing libraries with Nextera XT DNA Library Prep kit at half volume, as follows.
SECTION: Library Preparation
11. Tagment DNA. 

Thaw the following Nextera XT reagents on ice: 

Amplicon tagment mix (ATM)
Tagment DNA buffer (TD)
Nextera PCR master mix (NPM)

Invert all reagents 3 - 5 times, followed by pulse spin. 

SECTION: Library Preparation
11.1. Add the following reagents in order:
5 µL of TD buffer
2.5 µL   of 0.2 ng/ul   amplicon (from step 9)
2.5 µL  of ATM

Cover plate with plate seal, mix gently on plate mixer and centrifuge for 1 min.

SECTION: Library Preparation
11.2. Incubate in thermocycler with the following steps:

Step    Temperature            Time                    Cycles
1            55 °C         5 min          1
2            10 °C           Hold

SECTION: Library Preparation
11.3. Remove the plate immediately once thermocycler reachs 10 °C  , and proceed to neutralization.
Add 2.5 µL of NT buffer to each well and mix by pipetting up and down for 3 times, briefly spin down the plate and incubate at room temperature   for 5 min.

SECTION: Library Preparation
12. PCR Amplification.

Thaw the following reagents on ice:
NPM
Index primers 
Resuspension buffer (RSB)

Thaw the index primers, mix by vortexing each vial and spin down the liquid at the bottom of the vials. Option to dispense indexes into 96 well plate for easier pipetting.
SECTION: Library Preparation
12.1. Add 7.5 µLof Nextera PCR mastermix to each well.

SECTION: Library Preparation
12.2. From the pre-aliquoted index plate, add 5 µL  (2.5 µL   of each i5 and i7 index)  of the corresponding index combination to each well. Cover plate with plate seal, gently mix on plate mixer, and centrifuge for 1 min.

SECTION: Library Preparation
12.3. Run the PCR program to amplify the libraries:

Step                        Temperature             Time                Cycles
1                           72 °C       3 min              1
2                           95 °C       30 s              1
3                           95 °C       10 s              12 
3                           55 °C       30 s              12
3                           72 °C       30 s              12
4                           72 °C       5 min               1
5                           4 °C          Hold                                1

SECTION: Library Cleanup
13. 
Repeat the same clean up process as step 6.1-6.8 using 20 µL   of AMPure XP beads and 28 µL   of resuspension buffer.

SECTION: Library Quantification
14. Repeat the same quantification process as Step 8 but do NOT dilute libraries.
SECTION: Normalization and Loading on Illumina Sequencing Instrument
15. Normalize each library to 4 nanomolar (nM) by dilution with nuclease free water. 

SECTION: Normalization and Loading on Illumina Sequencing Instrument
16. Pool equal volume (e.g. 5 µL ) from each of the normalized libraries into a single 1.5 mL  microtube. 

SECTION: Normalization and Loading on Illumina Sequencing Instrument
17. Verify fragment size and concentration using Agilent D5000 Assay on TapeStation 4200 as follows.
SECTION: Normalization and Loading on Illumina Sequencing Instrument
17.1. Add 2 µL of Sample Buffer and2 µL of your pooled libraries in triplicate in a strip tube. 

SECTION: Normalization and Loading on Illumina Sequencing Instrument
17.2. Vortex using the adapter at 2000 rpm for1 min  .

SECTION: Normalization and Loading on Illumina Sequencing Instrument
17.3. Load tubes, tapes, and tips into TapeStation. Start run. Using library concentration and fragment size, calculate the molarity of the libraries using the following formula:
Molarity = concentration ng/uL * (1515.1515/fragment size(bp))
SECTION: Normalization and Loading on Illumina Sequencing Instrument
18. Denature and load pooled libraries for MiSeq as follows.

NOTE: Remove sequencing kit components from freezer to thaw at appropriate temperature/time.
SECTION: Normalization and Loading on Illumina Sequencing Instrument
18.1. Denature the pooled libraries by mixing 5 µL   of pooled libraries and5 µL of freshly made 0.2N NaOH solution.
Incubate for 5 min.

SECTION: Normalization and Loading on Illumina Sequencing Instrument
18.2. Add 990 µL   of HT1 buffer and mix well with denatured pooled library by pipetting up and down 10 times with P1000. 

SECTION: Normalization and Loading on Illumina Sequencing Instrument
18.3. Load 600 µL  of the denatured, diluted pooled library into the loading position of the Illumina MiSeq reagent cartridge (V2, 300 cycle kit). Load reagent cartridge, flow cell, and PR2 buffer into Miseq instrument, confirm the metrics and start the run. 

SECTION: Normalization and Loading on Illumina Sequencing Instrument
19. For NextSeq loading, combine up to four pools of libraries at equal concentrations. Be careful to use unique index combinations for all pooled samples.

Denature and load pooled libraries for NextSeq as follows.


NOTE: Remove sequencing kit components from freezer to thaw at appropriate temperature/time.
SECTION: Normalization and Loading on Illumina Sequencing Instrument
19.1. Denature the pooled libraries by mixing 5 µL   of pooled libraries and5 µL of freshly made 0.2N NaOH solution.
Incubate for 5 min.
SECTION: Normalization and Loading on Illumina Sequencing Instrument
19.2. Add 5 µL 200 mM TrisHCl pH7.5 and 985 µL HT1 buffer, 

SECTION: Normalization and Loading on Illumina Sequencing Instrument
19.3. Pipette 97 µL of this denatured library solution into a new tube and add 1203 µL of HT1 buffer.

SECTION: Normalization and Loading on Illumina Sequencing Instrument
19.4. Load 1300 µLof the denatured, diluted pooled library into the loading position of the Illumina NextSeq reagent cartridge. Follow the prompts in the instrument to complete loading of flow cell, buffer bottle or cartridge and reagents cartridge