TITLE: SARS-CoV-2 Illumina MiSeq protocol v.1
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 by pipetting up and down 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 pippet to aliquot  of RNA to the plate from step 2, mix up and down 5x  by multichannel pippette, briefly centrifuge the plate to collect the liquid at bottom of the wells.
11 µl
?. Incubate the reaction mix in thermocycler as follows:       Hold
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. Mix gently by pipetting up and down and briefly centrifuge to collect liquid at the bottom of the well.
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]
Perform reverse transcription reaction in thermocycler as follows:            Hold
42 °C
70 °C
5 °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 and mix well by pipetting up and down. Seal the plates and briefly centrifuge to collect liquid at the bottom of the wells.
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
?. [Gel Electrophoresis]
Remove plates Pool 1 and Pool 2 amplicons from thermocyclers, briefly centrifuge.
?. [Gel Electrophoresis]
Prepare large 1% agarose gels with TBE buffer with enough wells for each PCR product and a ladder on each row.
?. [Gel Electrophoresis]
Mix  of 6X loading dye with  of PCR product and load gels with Pool 1 amplicons leaving a space between each sample to be filled by Pool 2 amplicons so that each sample can be visualized for both pools. Add 100bp ladder to left- and right-most wells.
2 µl
2 µl
?. [Gel Electrophoresis]
Confirm that presence/absence of bands in gels correlate RT-PCR Ct values for each sample (e.g. if band is absent, does sample have E-gene Ct >30?). Band sizes should be approximately 400bp.
?. [Gel Electrophoresis]
Run gels electrophoresis at 140V for
?. [Amplicon Clean-up]
Combine the two pools of amplicons into one: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]
Add   of AMPure XP beads(at  ) to the combined amplicons plate. Mix by pippetting up and down 10x, briefly centrifugre, and incubate for  .
25 µl
0 Room temperature
?. [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 and appropriate amount of nuclease free water.
2.5 µl
?. [Library preparation]
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 bottowm of the vials. Neutralization buffer (NT) at
0 Room temperature
?. [Library preparation]
Add the following reagents in order:  of TD buffer  of   amplicon   of ATMMix by pipetting up and down for 5 times.
5 µl
2.5 µl
5 µl
?. [Library preparation]
Seal plate and briefly centrifuge. 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]
Place the i5 and i7 index primers on the Illumina Index Adapter plate accordingly,Add  of NPM to each wells and mix by pipetting up and down for 3 times.Add  each of i5 and i7 index primers to each samples accordingly and mix by pipetting up and down for 5 times.Seal plate and briefly centrifuge.
7.5 µl
2.5 µl
?. [Library preparation]
Run the 1 hour PCR program to amplify the libraries:Step                        Temperature             Time                Cycles1                                           12                                           13                                           13 3                                           133                                           134                                           15                                           Hold                            1
72 °C
95 °C
95 °C
55 °C
72 °C
72 °C
4 °C
?. [Library preparation]
Briefly spin the plate to collect liquid at the bottom of the wells.Repeat the same clean up process as step 16 using   of AMPure XP beads and   of resuspension buffer (step 16.8).
20 µl
28 µl
?. [Normalization and loading on Miseq]
Verify fragment size using Agilent D5000 Assay on TapeStation 4200.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]
Normalize all the libraries to  by dilution with nuclease free water.
?. [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. Further dilute the pool library down to   with HT1 buffer to prevent over clusering, if the HS D1000 tape station concentration of the library shows greater than 4nM.
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
?. [Amplicon Clean-up]
Place the plate on a magnetic rack for  , or until the beads have pelleted and the supernatant is completely clear.
?. [Amplicon Clean-up]
Remove and discard the liquid from each well with a multichannel pippette.
?. [Amplicon Clean-up]
Add   of freshly prepared 80% ethanol to each well, incubate for  , remove the ethanol carefully with a multichannel pippette.
200 µl
?. [Amplicon Clean-up]
Repeat ethanol wash (Step 16.3).
?. [Amplicon Clean-up]
Remove the residual ethanol with a P20 multichannel pippette, allow plate to dry for 3-5 minutes on the magnetic rack or until the pellet loses its shine (do not over-dry the pellet).
?. [Amplicon Clean-up]
Remove from magnetic rack, add   of EB buffer to wells and mix thoroughly by multichannel pippetting, until beads are resuspended. 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]
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]
Transfer   of the clear supernatant to a new plate, ensuring no beads are transferred.
25 µl
?. [Amplicon quantification and normalization]
Quantify amplicons using Qubit dsDNA High Sensitivity and DTX880 plate reader.
?. [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.
[X]
[X]
98 µl
?. [Amplicon quantification and normalization]
Dilute the amplicon (from step 16.8) 1:10 by mixing   of the amplicon 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 DTX880 plate reader to obtain reading for each sample and standards. The Qubit standard curve is generated by the Qubit standards.
2 µl
98 µl
?. [Library preparation]
Repeat the same quantification process as Step 17 but do NOT dilute libraries.
?. [Normalization and loading on Miseq]
Pool   from each of the normalized libraries into a signal  microtube. Measure the final pool concentration in duplicate using HS D1000 tape station.
5 µl
1.5 mL