Jun 23, 2022
Version 2
nCoV-2019 McGill Artic PCR Protocol, V4.1 at 63C V.2
Version 1 is forked from nCoV-2019 McGill Artic PCR Protocol, 5 ul RT and V3 only + LA1 at 63C
- 1McGill University
Protocol Citation: Sarah J Reiling, Kayleigh Loranger, Anne-Marie Roy, Shu-Huang Chen, Ioannis Ragoussis 2022. nCoV-2019 McGill Artic PCR Protocol, V4.1 at 63C. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov18e4ygr2/v2Version created by Kayleigh Loranger
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: June 23, 2022
Last Modified: June 23, 2022
Protocol Integer ID: 65186
Abstract
This is the updated SARS-Cov-2 PCR Protocol, with the ARTIC V4.1 primers, that is currently being used at the McGill Genome Center.
Materials
MATERIALS
Q5 High-Fidelity 2X Master Mix - 500 rxnsNew England BiolabsCatalog #M0492L
nuclease-free water
Fresh 80% Ethanol
Quant-iT™ PicoGreen™ dsDNA Assay KitInvitrogen - Thermo FisherCatalog #P11496
AmpureXP beads Beckman CoulterCatalog #A63880
Primer pool preparation
Primer pool preparation
PRIMER POOL PREPARATION
If required resuspend lyophilised primers at a concentration of 100 µM each
Note
V4.1 only primers for this protocol were designed using Primal Scheme and generate overlapping 400 nt amplicons. V4.1 was added to the V4 primer set for optimization. Primer names and dilutions are listed below.
For information on V4 primers visit Optimization of the SARS-CoV-2 ARTIC Network V4 Primers and Whole Genome Sequencing Protocol - PMC (nih.gov)
The V4.1 pre-pooled primers in 1.5 mL Eppendorf labelled tubes are labelled “Pool 1 (100µM)” or “Pool 2 (100µM)". The primers do not require additional preparation.
Note
If the V4.1 primers are not pre-pooled, follow the below pipetting scheme to make the master mix by adding the following primers to the V4 primer pools.
Added to pool 1:
SARS-CoV-2_23_RIGHT_alt1
SARS-CoV-2_27_RIGHT_alt1
SARS-CoV-2_79_RIGHT_alt1
SARS-CoV-2_89_LEFT_alt1
SARS-CoV-2_89_RIGHT_alt1
Added to pool 2:
SARS-CoV-2_10_LEFT_alt1
SARS-CoV-2_10_RIGHT_alt1
SARS-CoV-2_76_LEFT_alt1
SARS-CoV-2_76_RIGHT_alt1
SARS-CoV-2_88_LEFT_alt1
SARS-CoV-2_90_RIGHT_alt1
The guide to pooling volumes are as follows;
2x volume:
SARS-CoV-2_1_LEFT & SARS-CoV-2_1_RIGHT
SARS-CoV-2_7_LEFT & SARS-CoV-2_7_RIGHT
SARS-CoV-2_13_LEFT & SARS-CoV-2_13_RIGHT
SARS-CoV-2_17_LEFT & SARS-CoV-2_17_RIGHT
SARS-CoV-2_27_LEFT & SARS-CoV-2_27_RIGHT
SARS-CoV-2_45_LEFT & SARS-CoV-2_45_RIGHT
SARS-CoV-2_59_LEFT & SARS-CoV-2_59_RIGHT
SARS-CoV-2_60_LEFT & SARS-CoV-2_60_RIGHT
SARS-CoV-2_61_LEFT & SARS-CoV-2_61_RIGHT
SARS-CoV-2_64_LEFT & SARS-CoV-2_64_RIGHT
SARS-CoV-2_79_LEFT & SARS-CoV-2_79_RIGHT
SARS-CoV-2_90_LEFT & SARS-CoV-2_90_RIGHT
SARS-CoV-2_91_LEFT & SARS-CoV-2_91_RIGHT
1x volume: All other primers including alts (from V4.1).
Primers should be pooled in the mastermix cabinet which should be cleaned with decontamination wipes and UV sterilised before and after use.
Multiplex PCR
Multiplex PCR
MULTIPLEX PCR
In the extraction and sample addition cabinet add 5 µL RT product to each tube and mix well by pipetting.
Note
The extraction and sample addition cabinet should should be cleaned with decontamination wipes and UV sterilised before and after use.
In the mastermix hood set up the multiplex PCR reactions as follows in 0.2mL 8-strip PCR tubes:
Component Pool 1 [10 uM primer] Pool 2 [10 uM]
Q5 Hot Start High-Fidelity 2X Master Mix 12.5 µL 12.5 µL
Primer Pool 1 or 2 (10µM pool 1+2) 3.7 µL 3.7 µL
Nuclease-free water 3.8 µL 3.8 µL
Total 20 µL 20 µL
Add 20ul of PCR mastermix to the 5 ul RT product of step 10.
Note
A PCR mastermix for each pool should be made up in the mastermix cabinet and aliquoted into PCR strip tubes. Tubes should be wiped down when entering and leaving the mastermix cabinet.
Pulse centrifuge the tubes to collect the contents at the bottom of the tube.
Set-up the following program on the thermal cycler:
Step Temperature Time Cycles
Heat Activation 98 °C 00:00:30 1
Denaturation 98 °C 00:00:15 36
Annealing 63 °C 00:05:00 36
Hold 4 °C Indefinite 1
Note
Cycle number should be 25 for Ct 18-21 up to a maximum of 36 cycles for Ct 35
PCR clean-up
PCR clean-up
PCR CLEANUP
Combine the entire contents of “Pool 1” and “Pool 2” PCR reactions for each biological sample into to a single 1.5 mL Eppendorf tube.
Clean-up the amplicons using the following protocol:
Add an equal volume (1:1) of AmpureXP beads to the sample tube and mix by pipetting.
Incubate for 5 min at room temperature.
Pellet on magnet for 5 min. Remove supernatant.
Add 200 ul of 80% ethanol to the pellet and wash twice.
Let the beads dry for 3 min.
Add 30 ul elution buffer and resuspend the beads. Incubate for 3 minutes.
Pellet on magnet for 5 min. Remove and keep eluate (30 ul).
Note
Amplicon clean-up should be performed in the post-PCR cabinet which should should be cleaned with decontamination wipes and UV sterilised before and after use.
Amplicon Quantification and normalisation
Amplicon Quantification and normalisation
AMPLICON QUANTIFICATION AND NORMALIZATION
Quantify the amplicon pools using a fluorimetric dsDNA assay. (e.g: PicoGreen with a standard curve 0-200ng)
We expect following concentrations:
Pool 1+2 combined:
100-150 ng/ul for Ct 14-24
30-80 ng/ul for Ct 25-29
10-30 ng/ul for Ct 30-36
Nextera Flex Library Prep:
After quantification of Pool 1+2, take a new plate and add 150 ng of Pool 1+2 and add up with nuclease-free water to a total volume of 30 ul (= 5 ng/ul).
Nanopore Library Prep:
After quantification of Pool 1+2, take a new plate and add 200 ng of Pool 1+2 and add up with nuclease-free water to a total volume of 20 ul (= 10 ng/ul).