Feb 22, 2022
SARS-CoV-2 Amplicon-based Illumina Sequencing Protocol
- 1Public Health Ontario
Protocol Citation: wgscov 2022. SARS-CoV-2 Amplicon-based Illumina Sequencing Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.b5ftq3nn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: February 22, 2022
Last Modified: February 22, 2022
Protocol Integer ID: 58579
Abstract
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
Preamble to cDNA Synthesis
Preamble to cDNA Synthesis
Use either a SuperScript (step 2) or LunaScript kit (step 3) for cDNA synthesis
cDNA Synthesis
cDNA Synthesis
1h 30m
1h 30m
Option 1: SuperScipt mastermix
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.
Incubate the reaction in a thermocycler as follows:
Step Temperature Time Cycles
1 65 °C 00:05:00 1
2 4 °C Hold
5m
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.
Incubate in a thermocycler as follows:
Step Temperature Time Cycles
1 42 °C 00:50:00 1
2 70 °C 00:10:00 1
3 4 °C Hold
1h
Option 2: LunaScript mastermix
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.
Incubate the reaction in a thermocycler as follows:
Step Temperature Time Cycles
1 25 °C 00:02:00 1
2 55 °C 00:20:00 1
3 95 °C 00:01:00 1
4 4 °C 00:02:00 1
25m
Multiplex PCR Amplification
Multiplex PCR Amplification
5m 45s
5m 45s
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.
Run the 3.5 hours PCR program for each pool:
Step Temperature Time Cycles
Heat Activation 98 °C 00:00:30 1
Denaturation 98 °C 00:00:15 35
Annealing 65 °C 00:05:00 35
Hold 4 °C 1
5m 45s
Amplicon Pooling and Cleanup
Amplicon Pooling and Cleanup
22m 30s
22m 30s
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.
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 00:05:00 .
5m
Place the plate on a magnetic rack for 00:05:00 , or until the beads have pelleted and the supernatant is completely clear.
5m
Remove and discard the liquid from each well with a multichannel pippette, being careful not to touch the bead pellet.
Add 200 µL of freshly prepared, room temperature 80% ethanol to each well, incubate for 00:00:30 , remove the ethanol carefully with a multichannel pipette.
30s
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 00:03:00 to 00:05:00 or until the pellet loses its shine (if the pellet dries completely it will crack and become difficult to resuspend).
8m
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 00:05:00 .
5m
Place the plate on magnetic rack and incubate for 00:02:00 to 00:05:00 or until the beads have pelleted and the supernatant is completely clear.
7m
Transfer 25 µL of the clear supernatant to a new plate, ensuring no beads are transferred.
Gel Electrophoresis
Gel Electrophoresis
Optional section; use remaining volumes from Pool 1 and Pool 2 to confirm amplification by gel electrophoresis.
Prepare 1% agarose gels with enough wells to load all samples. Load 2 µL of a 100 bp ladder into gel on either side of each row of wells.
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.
Run at 240V for 00:20:00 . Visualize PCR products, confirm bands of approximately 300bp size.
20m
Amplicon Quantification and Normalization
Amplicon Quantification and Normalization
Quantify amplicons using Qubit dsDNA High Sensitivity kit and plate reader according to directions, as follows.
Create Qubit dsDNA HS working solution by mixing 99.5 µL X buffer and 0.5 µL X 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).
Dilute the clean, pooled amplicons (from step 6.8) 1:10 by mixing3 µL of the amplicons in 27 µL of nuclease free water.
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/ul plus Standard 1 0 ng/ul .
Mix2 µL of 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.
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.
Library Preparation
Library Preparation
Prepare sequencing libraries with Nextera XT DNA Library Prep kit at half volume, as follows.
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.
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 00:01:00 .
1m
Incubate in thermocycler with the following steps:
Step Temperature Time Cycles
1 55 °C 00:05:00 1
2 10 °C Hold
5m
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 00:05:00 .
5m
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.
Add 7.5 µL of Nextera PCR mastermix to each well.
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 00:01:00 .
1m
Run the PCR program to amplify the libraries:
Step Temperature Time Cycles
1 72 °C 00:03:00 1
2 95 °C 00:00:30 1
3 95 °C 00:00:10 12
3 55 °C 00:00:30 12
3 72 °C 00:00:30 12
4 72 °C 00:05:00 1
5 4 °C Hold 1
9m 40s
Library Cleanup
Library Cleanup
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.
Library Quantification
Library Quantification
Repeat the same quantification process as Step 8 but do NOT dilute libraries.
Normalization and Loading on Illumina Sequencing Instrument
Normalization and Loading on Illumina Sequencing Instrument
11m
11m
Normalize each library to 4 nanomolar (nM) by dilution with nuclease free water.
Pool equal volume (e.g. 5 µL ) from each of the normalized libraries into a single 1.5 mL microtube.
Verify fragment size and concentration using Agilent D5000 Assay on TapeStation 4200 as follows.
Add 2 µL of Sample Buffer and2 µL of your pooled libraries in triplicate in a strip tube.
Vortex using the adapter at 2000 rpm for00:01:00 .
1m
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))
Denature and load pooled libraries for MiSeq as follows.
NOTE: Remove sequencing kit components from freezer to thaw at appropriate temperature/time.
Denature the pooled libraries by mixing 5 µL of pooled libraries and5 µL of freshly made 0.2N NaOH solution.
Incubate for 00:05:00 .
5m
Add 990 µL of HT1 buffer and mix well with denatured pooled library by pipetting up and down 10 times with P1000.
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.
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.
Denature the pooled libraries by mixing 5 µL of pooled libraries and5 µL of freshly made 0.2N NaOH solution.
Incubate for 00:05:00 .
5m
Add 5 µL 200 mM TrisHCl pH7.5 and 985 µL HT1 buffer,
Pipette 97 µL of this denatured library solution into a new tube and add 1203 µL of HT1 buffer.
Load 1300 µL of 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