Aug 25, 2020

Public workspacenCoV-2019 sequencing protocol v3 (LoCost) V.3

Version 1 is forked from Ebola virus sequencing protocol
DOI
dx.doi.org/10.17504/protocols.io.bp2l6n26rgqe/v3
nCoV-2019 sequencing protocol v3 (LoCost)
  • 1University of Birmingham
  • 1 more workspace
Josh Quick
Open access
DOI: dx.doi.org/10.17504/protocols.io.bp2l6n26rgqe/v3
Protocol Citation: Josh Quick 2020. nCoV-2019 sequencing protocol v3 (LoCost). protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6n26rgqe/v3Version created by Josh Quick
Manuscript citation:
Improvements to the ARTIC multiplex PCR method for SARS-CoV-2 genome sequencing using nanopore. John R Tyson, et. al. bioRxiv 2020.09.04.283077; doi:https://doi.org/10.1101/2020.09.04.283077
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: July 01, 2020
Last Modified: August 25, 2020
Protocol Integer ID: 38778
Abstract
Amplicon sequencing protocol for SARS-CoV-2 v3 (LoCost)

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.

Changes in this version:

-Up to 95 samples per run with EXP-NBD196 native barcode kit
-Substitution of SuperScript IV for LunaScript RT SuperMix and reaction volume reduced to 10 uL.
-Substitution of Ultra II Ligation Module for Blunt/TA Ligase Master Mix and reaction volume reduced to 10 µL.
-Native barcode ligation reaction volume reduced to 10 uL.
-SFB wash volume reduced.
Materials

ComponentSupplierPart number
ARTIC nCoV-2019 V3 panel (100uM)IDTSee links below
LunaScript RT SuperMix KitNEBE3010
Q5 Hot Start HF Polymerase orNEBM0493
Q5 Hot Start High-Fidelity 2X Master MixNEBM0494
dNTP Solution Mix (10 mM ea.)NEBN0447
Nuclease-free water (100 mL)NEBB1500
NEBNext Ultra II End Repair/dA-tailing moduleNEBE7546
Blunt/TA Ligase Master MixNEBM0367
Native Barcoding Expansion Kit 1-12 and/orONTEXP-NBD104
Native Barcoding Expansion Kit 13-24 orONTEXP-NBD114
Native Barcoding Expansion Kit 96ONTEXP-NBD196
AMPure XP beadsBeckmanA63881
NEBNext Quick Ligation ModuleNEBE6056S
Sequencing Auxiliary VialsONTEXP-AUX001
Short Fragment Buffer Expansion KitONTEXP-SFB001
Qubit dsDNA HS Assay KitThermoQ32854
Flow Cell Priming KitONTEXP-FLP002
Flow Cell Wash Kit (optional)ONTEXP-WSH003
R9.4.1 flow cellsONTFLO-MIN106

IDT premixed ARTIC nCoV-2019 V3 panel or order oligos individually.
Before start
Prepare between 11 and 95 RNA samples plus 1 negative control using this protocol.
cDNA preparation
cDNA preparation
30m
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.
Note
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.

Note
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.

Mix the following components in a PCR strip-tubes/plate. Gently mix by pipetting and pulse spin the tube to collect liquid.

ComponentVolume
LunaScript RT SuperMix (5X) 2 µL
Template RNA 8 µL
Total10 µL

Note
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.

Note
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.

Incubate the reaction as follows:

Temperature25 °C for Duration00:02:00
Temperature55 °C for Duration00:10:00
Temperature95 °C for Duration00:01:00
Hold at Temperature4 °C
Primer pool preparation (optional)
Primer pool preparation (optional)
2h
If making up primer pools from individual oligos fully resuspend lyophilised oligos in 1xTE to a concentration of Concentration100 micromolar (µM) , vortex thoroughly and spin down.
Note
If using IDT ARTIC nCoV-2019 V3 Panel (Concentration100 micromolar (µM) ) skip to step 6.



Sort all odd regions primers into one or more tube racks. Add Amount5 µL of each odd region primer to a Amount1.5 mL Eppendorf tube labelled "Pool 1 (Concentration100 micromolar (µM) )". Repeat the process for all even region primers for Pool 2. These are your Concentration100 micromolar (µM) stocks of each primer pool.

Note
Primers should be diluted and pooled in the mastermix cabinet which should be cleaned with decontamination wipes and UV sterilised before and after use.

Note
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

Dilute Concentration100 micromolar (µM) pools 1:10 in molecular grade water, to generate Concentration10 micromolar (µM) primer stocks.

Note
Primers are used at a final concentration of Concentration15 nanomolar (nM) per primer. In this case V3 pools have 110 primers in pool 1 and 108 primers in pool 2. so the requirement is ~Amount4 µL primer pool (Concentration10 micromolar (µM) ) per Amount25 µL reaction.


Note
Make up multiple Amount100 µL aliquots of Concentration10 micromolar (µM) primer dilutions and freeze them in case of degradation or contamination.


Multiplex PCR
Multiplex PCR
4h
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.


ComponentReaction 1Reaction 2
5X Q5 Reaction Buffer5 µL5 µL
10 mM dNTPs0.5 µL0.5 µL
Q5 Hot Start DNA Polymerase0.25 µL0.25 µL
V3 Pool 1 (10µM)4 µL0 µL
V3 Pool 2 (10µM)0 µL4 µL
Nuclease-free water12.75 µL12.75 µL
Total22.5 µL22.5 µL
For M0493

or

ComponentReaction 1Reaction 2
Q5 Hot Start High-Fidelity 2X Master Mix12.5 µL12.5 µL
V3 Pool 1 (10µM)4 µL0 µL
V3 Pool 2 (10µM)0 µL4 µL
Nuclease-free water6 µL6 µL
Total22.5 µL22.5 µL
For M0494

Note
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 Amount2.5 µL for downstream steps.


Note
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

Add Amount2.5 µL 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.
Note
Up to Amount5 µL cDNA can be added to each PCR reaction (in place of nuclease-free water) to improve amplification of low titre samples. Using Amount5 µL cDNA will require a Amount20 µL cDNA reaction and may be more likely to cause inhibition so use cautiously.


Note
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.

Amount0 µL Set-up the following program on the thermal cycler:

Step Temperature Time Cycles

Heat Activation Temperature98 °C Duration00:00:30 1
Denaturation Temperature98 °C Duration00:00:15 25-35
Annealing Temperature65 °C Duration00:05:00 25-35
Hold Temperature4 °C Indefinite 1


Note
Cycle number should be 25 for Ct 18-21 up to a maximum of 35 cycles for Ct 35.


Note
Thermocycler calibration can vary instrument to instrument. If you see amplicon 64 dropout then decrease the annealing/extension temperature to Temperature63 °C . Denaturation temperature of Temperature95 °C can also be used and may slightly increase PCR yields.


PCR dilution
PCR dilution
10m
Label strip-tubes/plate and combine the following volumes of each PCR reaction for Amount10 µL each sample:

ComponentVolume
Pool 1 PCR reaction2.5 µL
Pool 2 PCR reaction2.5 µL
Nuclease-free water45 µL
Total50 µL

Note
The PCR post-clean up concentration is typically around Concentration100 Mass Percent . 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 Concentration10 Mass Percent then continue.


Note
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
Native barocoding
2h
Barcode the amplicon pools using the one-pot native barcoding approach.
Protocol
One-pot native barcoding of amplicons v3 (LoCost)
NAME
One-pot native barcoding of amplicons v3 (LoCost)
CREATED BY
Josh Quick

In a new PCR strip-tube/plate set up the following reaction for each sample:


ComponentVolume
PCR dilution from previous step3.3 µL
Ultra II End Prep Reaction Buffer1.2 µL
Ultra II End Prep Enzyme Mix0.5 µL
Nuclease-free water5 µL
Total10 µL

Note
Make a master mix of end-preparation reagents and nuclease-free water and aliquot into strip-tube/plate to improve reproducability.


Incubate at room temperature for Duration00:15:00
Incubate at Temperature65 °C f for Duration00:15:00
Incubate on ice for Duration00:01:00

In a new PCR strip-tube/plate set up the following reaction for each sample:

ComponentVolume
End-preparation reaction mixture0.75 µL
NBXX barcode1.25 µL
Blunt/TA Ligase Master Mix5 µL
Nuclease-free water3 µL
Total10 µL

Note
Use one native barcode from the EXP-NBD104 (1-12), EXP-NBD114 (13-24) or EXP-NBD196 per sample. Use 12 or more barcodes per library or there will be insufficient total material to achieve good yields.


Incubate at room temperature for Duration00:20:00
Incubate at Temperature65 °C for Duration00:10:00
Incubate on ice for Duration00:01:00
Note
The 65°C incubation is to inactivate the DNA ligase to prevent barcode cross-ligation when reactions are pooled in the next step.

In a new Amount1.5 mL Eppendorf tube pool all one-pot barcoding reactions together.
Note
If processing 12-24 samples pool all Amount10 µL from each native barcoding reaction.
if processing 48 samples pool Amount5 µL from each native barcoding reaction.
If processing 96 samples pool Amount2.5 µL from each native barcoding reaction so as not to exceed a pool volume of Amount240 µL which would make the clean-up volume too large.




Add 0.4x volume of SPRI beads to the sample tube and mix gently by either flicking or pipetting. For example add Amount96 µL SPRI beads to Amount240 µL pooled one-pot barcoding reactions.

Note
0.4x volume of SPRI is sufficient to bind 400 bp amplicons in the presence of ligation buffer, do not use 1x as this will result in an excessive large bead pellet.

Mix by vortexing and pulse centrifuge to collect all liquid at the bottom of the tube. Incubate for Duration00:05:00 at room temperature.
Place on magnetic rack and incubate for Duration00:02:00 or until the beads have pelleted and the supernatant is completely clear. Carefully remove and discard the supernatant, being careful not to touch the bead pellet.

Add Amount250 µL SFB and resuspend beads completely by pipette mixing. Pulse centrifuge to collect all liquid at the bottom of the tube and place on the magnet. Remove supernatant and discard.
Note
SFB will remove excess adapter without damaging the adapter-protein complexes. Do not use 70% ethanol as in early clean-ups.



Repeat steps 11.9 to perform a second SFB wash. Pulse centrifuge and remove any residual SFB.
Note
You do not need to allow to air dry with SFB washes.

Add Amount200 µL of room-temperature Concentration70 % volume ethanol to bathe the pellet. Carefully remove and discard ethanol, being careful not to touch the bead pellet.
Note
Only perform 1x 70% ethanol wash



Pulse centrifuge to collect all liquid at the bottom of the tube and carefully remove as much residual ethanol as possible using a P10 pipette.
With the tube lid open incubate for Duration00:01:00 or until the pellet loses it's shine (if the pellet dries completely it will crack and become difficult to resuspend).

Resuspend pellet in Amount30 µL Concentration10 millimolar (mM) Tris pH 8.0, mix gently by either flicking or pipetting and incubate for Duration00:02:00 .

Place on magnet and transfer sample to a clean Amount1.5 mL Eppendorf tube ensuring no beads are transferred into this tube.

Quantify Amount1 µL 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 Amount30 ng total at this stage to acheive maximum run yield.
Protocol
DNA quantification using the Quantus fluorometer
NAME

DNA quantification using the Quantus fluorometer

CREATED BY
Josh Quick


Remove Lambda DNA 400 ng/µL standard from the freezer and leave on ice to thaw. Remove ONE dsDNA dye solution from the fridge and allow to come to room temperature.

ReagentQuantiFluor(R) ONE dsDNA System, 500rxnPromegaCatalog #E4870



Set up two Amount0.5 mL tubes for the calibration and label them 'Blank' and 'Standard'

Add Amount200 µL ONE dsDNA Dye solution to each tube.
Mix the Lambda DNA standard 400 ng/µL standard by pipetting then add Amount1 µL to one of the standard tube.

Mix each sample vigorously by vortexing for Duration00:00:05 and pulse centrifuge to collect the liquid.
Allow both tubes to incubate at room temperature for Duration00:02:00 before proceeding.

Selection 'Calibrate' then 'ONE DNA' then place the blank sample in the reader then select 'Read Blank'. Now place the standard in the reader and select 'Read Std'.
Set up the required number of Amount0.5 mL tubes for the number of DNA samples to be quantified.
Note
Use only thin-wall, clear, 0.5mL PCR tubes such as Axygen #PCR-05-C


Label the tubes on the lids, avoid marking the sides of the tube as this could interfere with the sample reading.
Add Amount199 µL ONE dsDNA dye solution to each tube.

Add Amount1 µL of each user sample to the appropriate tube.
Note
Use a P2 pipette for highest accuracy.


Mix each sample vigorously by vortexing for Duration00:00:05 and pulse centrifuge to collect the liquid.

Allow all tubes to incubate at room temperature forDuration00:02:00 before proceeding.

On the Home screen of the Quantus Fluorometer, select `Protocol`, then select `ONE DNA` as the assay type.
Note
If you have already performed a calibration for the selected assay you can continue, there is no need to perform repeat calibrations when using ONE DNA pre diluted dye solution. If you want to use the previous calibration, skip to step 11. Otherwise, continue with step 9.

On the home screen navigate to 'Sample Volume' and set it to Amount1 µL then 'Units' and set it to ng/µL.

Load the first sample into the reader and close the lid. The sample concentration is automatically read when you close the lid.
Repeat step 16 until all samples have been read.
The value displayed on the screen is the dsDNA concentration in ng/µL, carefully record all results in a spreadsheet or laboratory notebook.
Set up the following AMII adapter ligation and clean-up with SFB.

Protocol
Adapter ligation with AMII v2
NAME
Adapter ligation with AMII v2
CREATED BY
Josh Quick

In a new Amount1.5 µL Eppendorf tube set up the following AMII adapter ligation reaction.


ComponentVolume
Barcoded amplicon pool30 µL
NEBNext Quick Ligation Reaction Buffer (5X)10 µL
Adapter Mix (AMII)5 µL
Quick T4 DNA Ligase5 µL
Total50 µL


Incubate at room temperature for Duration00:20:00
Add Amount50 µL (1:1) of SPRI beads to the sample tube and mix gently by either flicking or pipetting. Pulse centrifuge to collect all liquid at the bottom of the tube.
Note
Vortex SPRI beads thoroughly before use to ensure they are well resuspended, the solution should be a homogenous brown colour.

Note
There will be some variation in clean-up efficiencies but expect to carry around 50% through this clean-up

Incubate for Duration00:05:00 at room temperature.

Place on magnetic rack and incubate for Duration00:02:00 or until the beads have pelleted and the supernatant is completely clear. Carefully remove and discard the supernatant, being careful not to touch the bead pellet.

Add Amount250 µL SFB and resuspend beads completely by pipette mixing. Pulse centrifuge to collect all liquid at the bottom of the tube. Remove supernatant and discard.

Note
SFB will remove excess adapter without damaging the adapter-protein complexes. Do not use 70% ethanol as in early clean-ups.

Repeat steps 13.6 to perform a second SFB wash.
Pulse centrifuge and remove any residual SFB. Add Amount15 µL EB (ONT) and resuspend beads by pipette mixing.
Note
You do not need to allow to air dry with SFB washes.

Incubate at room temperature for Duration00:02:00 .

Place on magnetic rack until clear. Transfer final library to a new 1.5mL Eppendorf tube.
Quantify Amount1 µL of the final library using the Quantus Fluorometer using the ONE dsDNA assay. Concentration will vary depending on number and Ct of samples but Amount15 ng final library is usually required to acheive maximum run yield.
Protocol
DNA quantification using the Quantus fluorometer
NAME

DNA quantification using the Quantus fluorometer

CREATED BY
Josh Quick

Note
Final library can be now be stored in Concentration10 millimolar (mM) Tris Ph8 at Temperature4 °C for up to a week if needed otherwise proceed directly to MinION sequencing.



Remove Lambda DNA 400 ng/µL standard from the freezer and leave on ice to thaw. Remove ONE dsDNA dye solution from the fridge and allow to come to room temperature.

ReagentQuantiFluor(R) ONE dsDNA System, 500rxnPromegaCatalog #E4870



Set up two Amount0.5 mL tubes for the calibration and label them 'Blank' and 'Standard'

Add Amount200 µL ONE dsDNA Dye solution to each tube.
Mix the Lambda DNA standard 400 ng/µL standard by pipetting then add Amount1 µL to one of the standard tube.

Mix each sample vigorously by vortexing for Duration00:00:05 and pulse centrifuge to collect the liquid.
Allow both tubes to incubate at room temperature for Duration00:02:00 before proceeding.

Selection 'Calibrate' then 'ONE DNA' then place the blank sample in the reader then select 'Read Blank'. Now place the standard in the reader and select 'Read Std'.
Set up the required number of Amount0.5 mL tubes for the number of DNA samples to be quantified.
Note
Use only thin-wall, clear, 0.5mL PCR tubes such as Axygen #PCR-05-C


Label the tubes on the lids, avoid marking the sides of the tube as this could interfere with the sample reading.
Add Amount199 µL ONE dsDNA dye solution to each tube.

Add Amount1 µL of each user sample to the appropriate tube.
Note
Use a P2 pipette for highest accuracy.


Mix each sample vigorously by vortexing for Duration00:00:05 and pulse centrifuge to collect the liquid.

Allow all tubes to incubate at room temperature forDuration00:02:00 before proceeding.

On the Home screen of the Quantus Fluorometer, select `Protocol`, then select `ONE DNA` as the assay type.
Note
If you have already performed a calibration for the selected assay you can continue, there is no need to perform repeat calibrations when using ONE DNA pre diluted dye solution. If you want to use the previous calibration, skip to step 11. Otherwise, continue with step 9.

On the home screen navigate to 'Sample Volume' and set it to Amount1 µL then 'Units' and set it to ng/µL.

Load the first sample into the reader and close the lid. The sample concentration is automatically read when you close the lid.
Repeat step 16 until all samples have been read.
The value displayed on the screen is the dsDNA concentration in ng/µL, carefully record all results in a spreadsheet or laboratory notebook.
MinION sequencing
MinION sequencing
1d
Prime the flowcell and load Amount15 ng sequencing library onto the flowcell.


Protocol
Priming and loading a MinION flowcell v2
NAME
Priming and loading a MinION flowcell v2
CREATED BY
Josh Quick

Note
From experience we know Amount15 ng is optimum loading input for short amplicons. Speed drop during the run indicates excessive library was loaded. Low run yield <20M reads indicates insufficient library.


Thaw the following reagents at room temperature before placing on ice:

Sequencing buffer (SQB)
Loading beads (LB)
Flush buffer (FLB)
Flush tether (FLT)
Add Amount30 µL FLT to the FLB tube and mix well by vortexing.

If required place a new MinION flowcell onto the MinION by flipping open the lip and pushing one end of the flowcell under the clip and pushing down gently.

Rotate the inlet port cover clockwise by 90° so that the priming port is visible.
Take a P1000 pipette and tip and set the volume to Amount800 µL . Place the tip in the inlet port and holding perpendicularly to the plane of the flowell remove any air from the inlet port by turning the volume dial anti-clockwise.
Note
Be careful not to remove so much volume that air is introduced onto the rectangular array via the outlet.


Load Amount800 µL of FLB (plus FLT) into the flow cell via the inlet port, dispense slowly and smoothly trying to avoid the introduction of any air bubbles.

Wait forDuration00:05:00 .

Gently lift the SpotON cover to open the SpotON port.
Load another Amount200 µL of FLB (plus FLT) into the flow cell via the inlet port, this will initiate a siphon at the SpotON port to allow you to load the library dilution.
In a new tube prepare the library dilution for sequencing:


ComponentVolume
SQB37.5 µL
LB25.5 µL
Library12 µL
Total75 µL


Note
Mix LB immediately before use as they settle quickly.

Make up with EB if less than 12 µL library is required.

Mix the prepared library gently by pipetting up and down just prior to loading.
Add the Amount75 µL library dilution to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop siphons into the port before adding the next.

Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the inlet port and close the MinION lid.
Start the sequencing run using MinKNOW.

Protocol
Starting a MinION sequencing run using MinKNOW
NAME
Starting a MinION sequencing run using MinKNOW
CREATED BY
Josh Quick

Note
If using Live basecalling ensure to turn on double-ended barcoding in the basecalling settings.



If required plug the MinION into the computer and wait for the MinION and flowcell to ben detected.
Choose flow cell 'FLO-MIN106' from the drop-down menu.
Then select the flowcell so a tick appears.
Click the 'New Experiment' button in the bottom left of the screen.
On the New experiment popup screen, select the running parameters for your experiment from the individual tabs:

Experiment: Name the run in the experiment field, leave the sample field blank.

Kit: Selection: Select LSK109 as there is no option for native barcoding (NBD104).

Run Options: Set the run length to 6 hours (you can stop the run once sufficient data has been collected as determined using RAMPART).

Basecalling: Leave basecalling turned but select 'fast basecalling'.

Output: The number of files that MinKNOW will write to a single folder. By default this is set to 4000 but can be reduced to make RAMPART update more frequently.

Click 'Start run'.
Monitor the progress of the run using the MinKNOW interface.