TITLE: nCoV-2019 sequencing protocol v3 (LoCost)
AUTHORS: Josh Quick

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Amplicon sequencing protocol for SARS-CoV-2 v3 (LoCost)</div><div class = "text-block">We thank the ARTIC network, Oxford Nanopore Technologies, New England Biolabs, BCCDC, COG-UK, CanCOGen and protocols.io commenters for their assistance developing this protocol.</div><div class = "text-block">Changes in this version:</div><div class = "text-block">-Up to 95 samples per run with EXP-NBD196 native barcode kit</div><div class = "text-block">-Substitution of SuperScript IV for LunaScript RT SuperMix and reaction volume reduced to 10 uL.</div><div class = "text-block">-Substitution of Ultra II Ligation Module for Blunt/TA Ligase Master Mix and reaction volume reduced to 10 µL.</div><div class = "text-block">-Native barcode ligation reaction volume reduced to 10 uL.</div><div class = "text-block">-SFB wash volume reduced.</div></div>

[STEPS]
?. [cDNA preparation]
Mix the following components in a PCR strip-tubes/plate. Gently mix by pipetting and pulse spin the tube to collect liquid.  AB1ComponentVolume2LunaScript RT SuperMix (5X)   2 µL3Template RNA  8 µL4Total10 µL
AB1ComponentVolume2LunaScript RT SuperMix (5X)   2 µL3Template RNA  8 µL4Total10 µL
Viral RNA input from a clinical sample should be between Ct 18-35. If Ct is between 12-15, then dilute the sample 100-fold in water, if between 15-18 then dilute 10-fold in water. This will reduce the likelihood of PCR-inhibition.
To prevent pre-PCR contamination the mastermix should be added to the PCR strip-tubes/plate in the mastermix cabinet which should should be cleaned with decontamination wipes and UV sterilised before and after use. RNA samples should be added in the extraction/sample addition cabinet which should should be cleaned with decontamination wipes and UV sterilised before and after use.
?. [cDNA preparation]
Incubate the reaction as follows: for  for  for Hold at
25 °C
55 °C
95 °C
4 °C
?. [Multiplex PCR]
Set up the two PCR reactions per sample as follows in strip-tubes or plates. Gently mix by pipetting and pulse spin the tube to collect liquid at the bottom of the tube.  ABC1ComponentReaction 1Reaction 225X Q5 Reaction Buffer5 µL5 µL310 mM dNTPs0.5 µL0.5 µL4Q5 Hot Start DNA Polymerase0.25 µL0.25 µL5V3 Pool 1 (10µM)4 µL0 µL6V3 Pool 2 (10µM)0 µL4 µL7Nuclease-free water12.75 µL12.75 µL8Total22.5 µL22.5 µLor  ABC1ComponentReaction 1Reaction 22Q5 Hot Start High-Fidelity 2X Master Mix12.5 µL12.5 µL3V3 Pool 1 (10µM)4 µL0 µL4V3 Pool 2 (10µM)0 µL4 µL5Nuclease-free water6 µL6 µL6Total22.5 µL22.5 µL
ABC1ComponentReaction 1Reaction 225X Q5 Reaction Buffer5 µL5 µL310 mM dNTPs0.5 µL0.5 µL4Q5 Hot Start DNA Polymerase0.25 µL0.25 µL5V3 Pool 1 (10µM)4 µL0 µL6V3 Pool 2 (10µM)0 µL4 µL7Nuclease-free water12.75 µL12.75 µL8Total22.5 µL22.5 µL
ABC1ComponentReaction 1Reaction 22Q5 Hot Start High-Fidelity 2X Master Mix12.5 µL12.5 µL3V3 Pool 1 (10µM)4 µL0 µL4V3 Pool 2 (10µM)0 µL4 µL5Nuclease-free water6 µL6 µL6Total22.5 µL22.5 µL
Q5 Hot Start High-Fidelity 2X Master Mix can also be used instead of the component kit. Half-scale PCR reactions can also be used to save costs as you will only require   for downstream steps.
To prevent pre-PCR contamination the mastermix for each pool should be made up in the mastermix cabinet which should should be cleaned with decontamination wipes and UV sterilised before and after use and aliquoted into PCR strip-tubes/plate
?. [Multiplex PCR]
Add  cDNA to each of the PCR reactions, gently mix by pipetting and pulse spin the tube to collect liquid at the bottom of the tube.
2.5 µl
Up to    cDNA can be added to each PCR reaction (in place of nuclease-free water) to improve amplification of low titre samples. Using    cDNA will require a    cDNA reaction and may be more likely to cause inhibition so use cautiously.
cDNA should be added in the extraction and sample addition cabinet which should should be cleaned with decontamination wipes and UV sterilised before and after use.
?. [Multiplex PCR]
Set-up the following program on the thermal cycler:  Step                        Temperature     Time                    CyclesHeat Activation         1Denaturation              25-35 Annealing                   25-35Hold                                   Indefinite              1
0 µl
98 °C
98 °C
65 °C
4 °C
Cycle number should be 25 for Ct 18-21 up to a maximum of 35 cycles for Ct 35.
Thermocycler calibration can vary instrument to instrument. If you see amplicon 64 dropout then decrease the annealing/extension temperature to  . Denaturation temperature of   can also be used and may slightly increase PCR yields.
?. [PCR dilution]
Label strip-tubes/plate and combine the following volumes of each PCR reaction for  each sample:  AB1ComponentVolume2Pool 1 PCR reaction2.5 µL3Pool 2 PCR reaction2.5 µL4Nuclease-free water45 µL5Total50 µL
10 µl
AB1ComponentVolume2Pool 1 PCR reaction2.5 µL3Pool 2 PCR reaction2.5 µL4Nuclease-free water45 µL5Total50 µL
The PCR post-clean up concentration is typically around  . This means we can pool them without quantification/normalisation to make a significant time saving. If you require very even barcode representation perform clean-up and normalise to   then continue.
Amplicons should be added in the post-PCR cabinet which should should be cleaned with decontamination wipes and UV sterilised before and after use.
?. [Native barocoding]
Barcode the amplicon pools using the one-pot native barcoding approach.
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?. [Native barocoding]
Quantify  of the barcoded amplicons using the Quantus Fluorometer using the ONE dsDNA assay. Concentration will vary depending on number and Ct of samples and but you need about   total at this stage to acheive maximum run yield.
1 µl
30 ng
?. [Native barocoding]
Set up the following AMII adapter ligation and clean-up with SFB.
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?. [Native barocoding]
Quantify  of the final library using the Quantus Fluorometer using the ONE dsDNA assay. Concentration will vary depending on number and Ct of samples but  final library is usually required to acheive maximum run yield.
1 µl
15 ng
Final library can be now be stored in   Tris    at    for up to a week if needed otherwise proceed directly to MinION sequencing.
?. [MinION sequencing]
Prime the flowcell and load  sequencing library onto the flowcell.
15 ng
From experience we know    is optimum loading input for short amplicons. Speed drop during the run indicates excessive library was loaded. Low run yield
?. [MinION sequencing]
Start the sequencing run using MinKNOW.
If using Live basecalling ensure to turn on double-ended barcoding in the basecalling settings.
?. [Primer pool preparation (optional)]
If making up primer pools from individual oligos fully resuspend lyophilised oligos in 1xTE to a concentration of , vortex thoroughly and spin down.
If using IDT ARTIC nCoV-2019 V3 Panel (  ) skip to step 6.
?. [Primer pool preparation (optional)]
Dilute   pools 1:10 in molecular grade water, to generate  primer stocks.
Primers are used at a final concentration of  per primer. In this case V3 pools have 110 primers in pool 1 and 108 primers in pool 2. so the requirement is ~  primer pool ( ) per   reaction.
Make up multiple   aliquots of  primer dilutions and freeze them in case of degradation or contamination.
?. [Primer pool preparation (optional)]
Sort all odd regions primers into one or more tube racks. Add  of each odd region primer to a Eppendorf tube labelled "Pool 1 ()". Repeat the process for all even region primers for Pool 2. These are your  stocks of each primer pool.
5 µl
1.5 mL
Primers should be diluted and pooled in the mastermix cabinet which should be cleaned with decontamination wipes and UV sterilised before and after use.
For more information see Figure 2 in;Quick, J. et al. Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples. Nat Protoc 12, 1261–1276 (2017). https://doi.org/10.1038/nprot.2017.066
?. [cDNA preparation]
Prepare between 11 and 95 RNA samples plus 1 negative control of nuclease-free water per library. If previously frozen, mix by briefly vortexing and pulse spin to collect liquid. Keep samples on ice at all times.
At least 11 samples are required to have sufficient material to load on the sequencer at the end. If you process >23 you will need the EXP-NBD196 expansion kit.
A positive control can also be included which may be a synthetic RNA constructs or high-titre clinical sample which can be diluted. This can help monitor run performance.