TITLE: SARS-CoV-2 Illumina MiSeq protocol v.2
AUTHORS: Public Health Ontario

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>ARTIC amplicon sequencing protocol adapted from Josh Quick's </span><a href="https://www.protocols.io/view/ncov-2019-sequencing-protocol-v2-bdp7i5rn" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/view/ncov-2019-sequencing-protocol-v2-bdp7i5rn</span></a><span> for illumina sequencing of SARS-CoV-2</span></div></div>

[STEPS]
?. [cDNA preparation]
Mix the following components:Component                                                    Volume50 µM random hexamers                            10mM dNTPs mix (10mM each)               Total Mastermix volume                             (template RNA)                                            Total Reaction volume                                 Prepare Mastermix (1:1) of random hexamers and dNTP.Mix gently and pulse centrifuge to collect liquid at the bottom of the Mastermix tube.
1 µl
1 µl
2 µl
11 µl
13 µl
The Mastermix should be prepared in a clean room and the nucleic acids added in a BSC or workbench exclusive for RNA work.
?. [cDNA preparation]
Aliquot  of this mix into each well of a 96 well plate. Keep the plate in a cold block.
2 µl
?. [cDNA preparation]
Use multichannel pipette to aliquot  of RNA to the plate from step 2. Seal plate, mix gently on plate mixer, and briefly centrifuge the plate to collect the liquid at bottom of the wells.
11 µl
?. [cDNA preparation]
Incubate the reaction mix in thermocycler as follows:
65 °C
4 °C
?. [cDNA preparation]
Prepare the following mastermix: Component	                                        Volume SSIV Buffer	                                         100mM DTT                                         RNaseOUT RNase Inhibitor 	        SSIV Reverse Transcriptase	       Total Mastermix volume                    (denatured RNA)                                 Total Reaction volume                        Add  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.
4 µl
1 µl
1 µl
1 µl
7 µl
13 µl
20 µl
7 µl
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.
?. [cDNA preparation]
Incubate in a thermocycler as follows:            Hold
42 °C
70 °C
4 °C
?. [Multiplex PCR]
Prepare the multiplex PCR reactions as follows and aliquot in each well of a 96-well plate x2 (1 for each pool):Component                                                Pool 1                      Pool 2 5X Q5 Reaction Buffer                           10 mM dNTPs                                         Q5 Hot Start DNA Polymerase             Primer Pool 1 or 2 (10µM)                    Nuclease-free water                               Total  Mastermix volume                      (cDNA)                                                     Total reaction volume                           Prealiquot   of each mastermix(pool1 and pool2) to each plate (pool1 and pool2) accordingly.
5 µl
5 µl
0.5 µl
0.5 µl
0.25 µl
0.25 µl
3.6 µl
3.6 µl
13.15 µl
13.15 µl
22.5 µl
22.5 µl
2.5 µl
2.5 µl
25 µl
25 µl
22.5 µl
?. [Multiplex PCR]
In a BSC or workbench exclusive for RNA work, add  of cDNA from step 6 to each 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.
2.5 µl
?. [Multiplex PCR]
Run the 3.5 hours PCR program for each pool:Step                        Temperature             Time                CyclesHeat Activation                 1Denaturation                    35 Annealing                         35Hold                                                                         1
98 °C
98 °C
65 °C
4 °C
?. [Amplicon Clean-up]
Combine the two pools of amplicons:Add  of each Pool 1 and Pool 2 (total 25μl) in an 0.2 ml PCR plate (low binding plate).
12.5 µl
?. [Amplicon Clean-up]
Perform AMPure XP bead cleanup according to directions, as follows.
?. [Amplicon quantification and normalization]
Quantify amplicons using Qubit dsDNA High Sensitivity kit and plate reader according to directions, as follows.
?. [Amplicon quantification and normalization]
Create Qubit dsDNA HS working solution by mixing   buffer and  dye (X is the total number of samples, including 6 standards). Using a reservoir and multichannel pipette, dispense    into required number of wells of a Costar 3590 flat-bottom plate (or as appropriate for plate reader).
[X]
[X]
98 µl
?. [Amplicon quantification and normalization]
Dilute the clean, pooled amplicons (from step 11.9) 1:10 by mixing   of the amplicons in   of nuclease free water.
3 µl
27 µl
?. [Amplicon quantification and normalization]
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:  plus Standard 1  .
[standard 1]
?. [Amplicon quantification and normalization]
Mix  of diluted amplicons and each of the 6 standards  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.
2 µl
98 µl
?. [Amplicon quantification and normalization]
Based on the amplicon concentration, normalize of all the samples amplicon concentration to  .This can be done by adding  of diluted amplicon to a plate with prealiquoted, appropriate amount of nuclease free water.
2.5 µl
?. [Library preparation]
Prepare sequencing libraries with Nextera XT DNA Library Prep kit at half volume, as follows.
?. [Library preparation]
Tagment DNA. Thaw the following Nextera XT reagents on ice: Amplicon tagment mix (ATM)Tagment DNA buffer (TD)Nextera PCR master mix (NPM)Thaw the index primers, mix by vortex each vial and spin down the liquid at the bottom of the vials. Neutralization buffer (NT) at
0 Room temperature
?. [Library preparation]
PCR Amplification.Thaw the following reagents on ice:NMPIndex primers Invert all reagents 3 - 5 times, followed by pulse spin.
?. [Library Clean-up]
Clean Up LibrariesRepeat the same clean up process as step 11 using   of AMPure XP beads and   of resuspension buffer.
20 µl
28 µl
?. [Library Quantification]
Repeat the same quantification process as Step 16 but do NOT dilute libraries.
?. [Normalization and loading on Miseq]
Verify fragment size and concentration using Agilent D5000 Assay on TapeStation 4200 as follows.
?. [Normalization and loading on Miseq]
Normalize each library to  by dilution with nuclease free water.
?. [Normalization and loading on Miseq]
Pool equal volume (e.g.  ) from each of the normalized libraries into a single  microtube.
5 µl
1.5 mL
?. [Normalization and loading on Miseq]
Denature and load pooled libraries as follows.
?. [Amplicon Clean-up]
Add  of AMPure XP beads(well-vortexed and at ) to the combined amplicons 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  for  .
25 µl
0 Room temperature
0 Room temperature
?. [Amplicon Clean-up]
Transfer   of the clear supernatant to a new plate, ensuring no beads are transferred.
25 µl
?. [Amplicon Clean-up]
Place the plate on magnetic rack and incubate for  to  or until the beads have pelleted and the supernatant is completely clear.
?. [Amplicon Clean-up]
Remove from magnetic rack, add   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   for .
28 µl
0 Room temperature
?. [Amplicon Clean-up]
Discard all ethanol and carefully remove as much residual ethanol as possible using a multichannel pipette. With the plate uncovered, incubate for 3-5 min or until the pellet loses its shine (if the pellet dries completely it will crack and become difficult to resuspend).
?. [Amplicon Clean-up]
Repeat ethanol wash (step 11.3 and 11.4).
?. [Amplicon Clean-up]
Add  of freshly prepared,  80% ethanol to each well, incubate for , remove the ethanol carefully with a multichannel pipette.
200 µl
0 Room temperature
?. [Amplicon Clean-up]
Remove and discard the liquid from each well with a multichannel pippette, being careful not to touch the bead pellet.
?. [Amplicon Clean-up]
Place the plate on a magnetic rack for  , or until the beads have pelleted and the supernatant is completely clear.
?. [Gel electrophoresis]
Use remaining volumes from Pool 1 and Pool 2 to confirm amplification (step 9). Make 1% agarose gels with enough wells for all samples.
?. [Gel electrophoresis]
Load 2 l of the 100 bp ladder into gel on either side of each row of wells.
?. [Gel electrophoresis]
Dispense 2 l of 6X loading dye into each sample with a multichannel pipette, mix and load 2 l of this mix into the gel.
?. [Gel electrophoresis]
Run at 240V for . Visualize PCR products, confirm bands of approximately 300bp size.
?. [Library preparation]
Add the following reagents in order:  of TD buffer  of   amplicon (from step 17)  of ATMCover plate with plate seal, mix gently on plate mixer and centrifuge for 1 min.
5 µl
2.5 µl
2.5 µl
?. [Library preparation]
Incubate in thermocycler with the following steps:  hold
55 °C
10 °C
?. [Library preparation]
Remove the plate immediately once thermocycler reachs  , and proceed to neutralization.Add  of NT buffer to each well and mix by pipetting up and down for 3 times, briefly spin down the plate and incubate at   for  .
10 °C
2.5 µl
0 Room temperature
?. [Library preparation]
Add 7.5 μl of Nextera PCR mastermix to each well.
?. [Library preparation]
From the pre-aliquoted index plate, add  (  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.
5 µl
2.5 µl
?. [Library preparation]
Run the PCR program to amplify the libraries:Step                        Temperature             Time                Cycles1                                           12                                           13                                           12 3                                           123                                           124                                           15                                           Hold                            1
72 °C
95 °C
95 °C
55 °C
72 °C
72 °C
4 °C
?. [Normalization and loading on Miseq]
Add 2 μl of Sample Buffer and 2 μl of your pooled libraries in triplicate in a strip tube.
?. [Normalization and loading on Miseq]
Vortex using the adapter at 2000 rpm for 1 min.
?. [Normalization and loading on Miseq]
Load tubes, tapes, and tips. 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))
?. [Normalization and loading on Miseq]
Denature the pooled libraries by mixing   of pooled libraries and  of freshly made 0.2N NaOH solution.Incubate for .
5 µl
5 µl
?. [Normalization and loading on Miseq]
Add   of HT1 buffer and mix well with denatured pooled library by pipetting up and down 10 times with P1000.
990 µl
?. [Normalization and loading on Miseq]
Load  of the denatured, diluted pooled library into the loading position of the Illumina reagent cartridge (V2, 300 cycle kit). Load reagent cartridge, flow cell, and PR2 buffer into Miseq instrument, confirm the metrics and start the run.
600 µl