Apr 09, 2020

Public workspacenCoV-2019 sequencing protocol v2 (GunIt) V.2

Version 1 is forked from Ebola virus sequencing protocol
DOI
dx.doi.org/10.17504/protocols.io.bdp7i5rn
  • 1University of Birmingham
  • ARTIC
  • Coronavirus Method Development Community
Josh Quick
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DOI: dx.doi.org/10.17504/protocols.io.bdp7i5rn
External link: https://doi.org/10.1371/journal.pone.0243412
Protocol Citation: Josh Quick 2020. nCoV-2019 sequencing protocol v2 (GunIt). protocols.io https://dx.doi.org/10.17504/protocols.io.bdp7i5rn
Manuscript citation:
Cool K, Gaudreault NN, Morozov I, Trujillo JD, Meekins DA, McDowell C, Carossino M, Bold D, Kwon T, Balaraman V, Madden DW, Artiaga BL, Pogranichniy RM, Sosa GR, Henningson J, Wilson WC, Balasuriya UBR, García-Sastre A, Richt JA, Infection and transmission of SARS-CoV-2 and its alpha variant in pregnant white-tailed deer. bioRxiv doi: 10.1101/2021.08.15.456341
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: March 14, 2020
Last Modified: April 09, 2020
Protocol Integer ID: 34271
Abstract
ARTIC amplicon sequencing protocol for MinION for nCoV-2019

This one-pot native barcoding protocol was developed in conjunction with Oxford Nanopore Technologies, New England Biolabs and BCCDC.
Materials
Primers 25nm, desalted, ideally LabReady formulation from IDT nCoV-2019/V2
Extraction kits; Zymo Quick-RNA Viral Kit Zymo R1034 or
QIAamp Viral RNA Mini Qiagen 52904
SuperScript IV (50 rxn) Thermo 18090050
dNTP mix (10 mM each) Thermo R0192
Random Hexamers (50 µM) Thermo N8080127
RNase OUT (125 rxn) Thermo 10777019
Q5 Hot Start HF Polymerase NEB M0493S
NEBNext Ultra II End-prep NEB E7546S
NEBNext Quick Ligation Module NEB E6056S
Native Barcoding Expansion Kit 1-12 Nanopore EXP-NBD104
Native Barcoding Expansion Kit 13-24 Nanopore EXP-NBD114
Sequencing Auxiliary Vials Nanopore EXP-AUX001
Short Fragment Buffer Expansion kit Nanopore EXP-SFB001
Flow Cell Priming Kit Nanopore EXP-FLP002
R9.4.1 flow cells Nanopore FLO-MIN106
cDNA preparation
cDNA preparation
Mix the following components in an 0.2mL 8-strip tube;

Component Volume

50µM random hexamers Amount1 µL
10mM dNTPs mix (10mM each) Amount1 µL
Template RNA Amount11 µL
Total Amount13 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
A mastermix 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.

Gently mix by pipetting and pulse spin the tube to collect liquid at the bottom of the tube.
Incubate the reaction as follows:

Temperature65 °C for Duration00:05:00
Place on ice for Duration00:01:00
Add the following to the annealed template RNA:

Component Volume

SSIV Buffer Amount4 µL
100mM DTT Amount1 µL
RNaseOUT RNase Inhibitor Amount1 µL
SSIV Reverse Transcriptase Amount1 µL
Total Amount20 µL


Note
A mastermix should be made up in the mastermix cabinet and added to the denatured RNA in the extraction and sample addition cabinet. Tubes should be wiped down when entering and leaving the mastermix cabinet.

Gently mix by pipetting and pulse spin the tube to collect liquid at the bottom of the tube.
Incubate the reaction as follows:

Temperature42 °C Duration00:50:00
Temperature70 °C Duration00:10:00
Hold at Temperature5 °C

Primer pool preparation
Primer pool preparation
If required resuspend lyophilised primers at a concentration of 100µM each


Note
nCov-2019/V3 primers for this protocol were designed using Primal Scheme and generate overlapping 400nt amplicons. Primer names and dilutions are listed in the table below.





Generate primer pool stocks by adding Amount5 µL of each odd region primer to a Amount1.5 mL Eppendorf labelled “Pool 1 (100µM)” and each even region primer to a Amount1.5 mL Eppendorf labelled “Pool 2 (100µM)”. The pool is also given in the .TSV files in the primalscheme output. These are your 100µM 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.

Dilute this primer pool 1:10 in molecular grade water, to generate 10µM primer stocks. It is recommend that multiple aliquots of each primer pool are made to in case of degradation or contamination.
Note
Primers need to be used at a final concentration of 0.015µM per primer. In this case V3 pools have 110 primers in pool 1 and 108 primers in pool 2. so the requirement is ~4µL primer pool (10uM) per 25µL reaction. For other schemes, adjust the volume added appropriately.

Multiplex PCR
Multiplex PCR
In the mastermix hood set up the multiplex PCR reactions as follows in 0.2mL 8-strip PCR tubes:

Component Pool 1 Pool 2

5X Q5 Reaction Buffer Amount5 µL Amount5 µL
10 mM dNTPs Amount0.5 µL Amount0.5 µL
Q5 Hot Start DNA Polymerase Amount0.25 µL Amount0.25 µL
V3 Primer Pool 1 or 2 (10µM) Amount4.0 µL Amount4.0 µL
Nuclease-free water Amount12.75 µL Amount12.75 µL
Total Amount22.5 µL Amount22.5 µL

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.

In the extraction and sample addition cabinet add Amount2.5 µL cDNA 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.

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 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

PCR clean-up
PCR clean-up
Label a Amount1.5 mL Eppendorf tube for each sample and assemble the following PCR dilution for each sample:

Component Volume

Pool 1 PCR reaction Amount2.5 µL
Pool 2 PCR reaction Amount2.5 µL
Nuclease-free water Amount45 µL
Note
The PCR post-clean up concentration is typically around Concentration100 ng/µL . This assumption means we can make a significant saving in terms of protocol time for only a slight loss in barcode representation.


Quantification and normalisation
Quantification and normalisation
Label another Amount1.5 mL Eppendorf tube for each sample.
Note
Input to the one-pot native barcoding reaction is 50ng per sample. Process at least 6 samples plus one negative control per library in order to have sufficient material to load on the sequencer at the end.

Native barocoding
Native barocoding
Barcode the amplicon pools using the one-pot native barcoding approach.
Protocol
One-pot native barcoding of amplicons v2
NAME

One-pot native barcoding of amplicons v2

CREATED BY
Josh Quick


Set up the following reaction for each sample:

Component Volume

PCR dilution from previous step Amount5 µL
Nuclease-free water Amount7.5 µL
Ultra II End Prep Reaction Buffer Amount1.75 µL
Ultra II End Prep Enzyme Mix Amount0.75 µL
Total Amount15 µL

Incubate at room temperature for Duration00:10:00
Incubate at Temperature65 °C f for Duration00:10:00
Incubate on ice for Duration00:01:00

In a new 1.5mL Eppendorf tube set up the following reaction:

Component Volume

Previous reaction mixture Amount1.5 µL
Nuclease-free water Amount5.7 µL
NBXX barcode Amount2.5 µL
Ultra II Ligation Master Mix Amount10 µL
Ligation Enhancer Amount0.3 µL
Total Amount20 µL
Note
Use one native barcode from the EXP-NBD104 (1-12) or EXP-NBD114 (13-24) per sample. Use from 6 to 24 barcodes in a library, any fewer and 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 1.5 ml Eppendorf tube pool all Amount20 µL one-pot barcoding reactions together.


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 12-plex pooled one-pot native barcoding reactions.

Note
0.4x volume of SPRI will only bind 400 bp amplicons in the presence of ligation buffer as in a one-pot reaction, do not use 1x as this will result in excessive native barcode carryover.

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 700 µl SFB and resuspend beads completely by pipette mixing.
Note
SFB will remove excess adapter without damaging the adapter-protein complexes. Do not use 70% ethanol as in early clean-ups.

Pulse centrifuge to collect all liquid at the bottom of the tube.
Remove supernatant and discard.
Repeat steps 11-13 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 200 µl of room-temperature 70 % 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 00:01:00 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 Elution Buffer (EB), mix gently by either flicking or pipetting and incubate for Duration00:02:00 .

Place on magnet and transfer sample to a clean 1.5mL Eppendorf tube ensuring no beads are transferred into this tube.
Quantify Amount1 µL of the barcoded amplicon pool using the Quantus Fluorometer using the ONE dsDNA assay.
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
NAME
Adapter ligation with AMII
CREATED BY
Josh Quick

Set up the following AMII adapter ligation reaction:

Component Volume

End-repaired amplicon pools Amount30 µL
NEBNext Quick Ligation Reaction Buffer (5X) Amount10 µL
Adapter Mix (AMII) Amount5 µL
Quick T4 DNA Ligase Amount5 µL
Total Amount50 µL

Note
There will be some variation in clean-up efficiencies but expect to carry around 80% through a clean-up.

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.
Note
Vortex SPRI beads thoroughly before use to ensure they are well resuspended, the solution should be a homogenous brown colour.


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.

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

Pulse centrifuge to collect all liquid at the bottom of the tube.
Remove supernatant and discard.
Repeat steps 14-16 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 Amount15 µL EB and resuspend beads by pipette mixing.

Incubate at room temperature for Duration00:02:00 .

Place on magnetic rack.
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.
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 10 mM Tris pH 8 at 4°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
Prime the flowcell and load Amount20 ng sequencing library onto the flowcell.

Protocol
Priming and loading a MinION flowcell
NAME

Priming and loading a MinION flowcell

CREATED BY
Josh Quick

Note
From experience we know 20 ng is optimum loading input for short amplicons.

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:

Component Volume

SQB Amount37.5 µL
LB Amount25.5 µL
Final library Amount12 µL
Total Amount75 µL

Note
Mix LB immediately before use as they settle quickly.

Dilute library in EB if 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



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.