TITLE: nCoV-2019 McGill Artic PCR Protocol, 5 ul RT and V3 only at 63C
AUTHORS: Sarah Reiling, Anne-Marie Roy, Shu-Huang Chen, Ioannis Ragoussis

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">V3 only primers can be found here:</div><div class = "text-block"><a href="https://github.com/sarahreiling/artic-ncov2019/blob/master/primer_schemes/nCoV-2019/V3/nCoV-2019_V3only.scheme.bed" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://github.com/sarahreiling/artic-ncov2019/blob/master/</span></a><span>primer_schemes/nCoV-2019/V3/nCoV-2019_V3only.scheme.bed</span></div></div>

[STEPS]
?. [Primer pool preparation]
PRIMER POOL PREPARATIONIf required resuspend lyophilised primers at a concentration of 100 µM each
V3 only primers for this protocol were designed using Primal Scheme and generate overlapping 400 nt amplicons. Primer names and dilutions are listed in the table below. https://github.com/sarahreiling/artic-ncov2019/blob/master/primer_schemes/nCoV-2019/V3/nCoV-2019_V3only.scheme.bed
?. [Primer pool preparation]
Generate primer pool stocks by adding  of each primer pair to a  Eppendorf labelled either “Pool 1 (100µM)” or “Pool 2 (100µM)”. Total volume should be  for Pool 1 (100µM) and  for Pool 2 (100µM). These are your 100µM stocks of each primer pool.
5 µl
1.5 mL
490 µl
490 µl
Primers should be diluted and pooled in the mastermix cabinet which should be cleaned with decontamination wipes and UV sterilised before and after use.
?. [Primer pool preparation]
Dilute this primer pool 1:10 in molecular grade water, to generate 10 µM primer stocks. It is recommend that multiple aliquots of each primer pool are made to in case of degradation or contamination.
Primers need to be used at a final concentration of 0.015µM per primer. In this case both pools have 98 primers in so the requirement is 3.65µL primer pools (10uM) per 25µL reaction. For other schemes, adjust the volume added appropriately.
?. [Multiplex PCR]
MULTIPLEX PCRIn the extraction and sample addition cabinet add  RT product to each tube and mix well by pipetting.
5 µl
The extraction and sample addition cabinet should should be cleaned with decontamination wipes and UV sterilised before and after use.
?. [Multiplex PCR]
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             Primer Pool 1 or 2 (10µM pool 1+2)                      Nuclease-free water                                                 Total                                                                            Add 20ul of PCR mastermix to the 5 ul RT product of step 10.
12.5 µl
12.5 µl
3.7 µl
3.7 µl
3.8 µl
3.8 µl
20 µl
20 µl
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.
?. [Multiplex PCR]
Pulse centrifuge the tubes to collect the contents at the bottom of the tube.
?. [Multiplex PCR]
Set-up the following program on the thermal cycler:  Step                        Temperature     Time                    CyclesHeat Activation         1Denaturation              36 Annealing                   36Hold                                   Indefinite              1
98 °C
98 °C
63 °C
4 °C
Cycle number should be 25 for Ct 18-21 up to a maximum of 36 cycles for Ct 35
?. [PCR clean-up]
PCR CLEANUPCombine the entire contents of “Pool 1” and “Pool 2” PCR reactions for each biological sample into to a single Eppendorf tube.
1.5 mL
?. [PCR clean-up]
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).
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 NORMALIZATIONQuantify 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 14-2430-80 ng/ul for Ct 25-2910-30 ng/ul for Ct 30-36
?. [Amplicon Quantification and normalisation]
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).