May 05, 2020

Public workspaceLong reads nanopore sequencing to recover SARS-CoV-2 whole genome V.3

DOI
dx.doi.org/10.17504/protocols.io.bfy7jpzn
  • 1Laboratory of Respiratory Viruses and Measles, Oswaldo Cruz Institute, FIOCRUZ, Brazil;
  • 2University College London, United Kingdom
  • Coronavirus Method Development Community
Paola Resende
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DOI: dx.doi.org/10.17504/protocols.io.bfy7jpzn
Protocol Citation: Paola Resende 2020. Long reads nanopore sequencing to recover SARS-CoV-2 whole genome . protocols.io https://dx.doi.org/10.17504/protocols.io.bfy7jpzn
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: In development
We are still developing and optimizing this protocol.
Created: May 05, 2020
Last Modified: May 05, 2020
Protocol Integer ID: 36607
Keywords: nanopore, long reads, sequencing, genome, SARS-CoV-2 sequencing, RSV sequencing, Parainfluenza sequencing,
Abstract
This protocol describes step-by-step instructions for building long (~2kb) amplicon libraries to recover SARS-CoV-2 genomes using the nanopore sequencing.

It can be applied for sequencing on the MinION device or GridION.

This protocol was developed in collaboration with the Laboratory of Respiratory Viruses and Measles, Oswaldo Cruz Institute, FIOCRUZ, Brazil, and the two sequencing facilities at Pathogen Genomic Unit (PGU) and UCL Genomics, University College London (UCL), United Kingdom.

This document describes the manual steps to perform the protocol, but for further information about the automation of the protocol, please, contact Pathogen Genomics Unit (PGU) - UCL.
Manager: Rachel Williams
https://www.ucl.ac.uk/infection-immunity/pathogen-genomics-unit

This protocol is based on the amplicon tiling strategy described previously by Quick J et al 2017. However, we have applied this strategy to recover long reads (2kb), then some adjustments were performed in the protocol.
CITATION
Quick J, Grubaugh ND, Pullan ST, Claro IM, Smith AD, Gangavarapu K, Oliveira G, Robles-Sikisaka R, Rogers TF, Beutler NA, Burton DR, Lewis-Ximenez LL, de Jesus JG, Giovanetti M, Hill SC, Black A, Bedford T, Carroll MW, Nunes M, Alcantara LC Jr, Sabino EC, Baylis SA, Faria NR, Loose M, Simpson JT, Pybus OG, Andersen KG, Loman NJ (2017). Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples.. Nature protocols.
LINK
https://doi.org/10.1038/nprot.2017.066
If you have doubts, we are happy to help you to test or implement the protocol.
Materials
MATERIALS
ReagentNEBNext Ultra II End Repair/dA-Tailing Module - 96 rxnsNew England BiolabsCatalog #E7546L
ReagentNEBNext Ultra II Ligation Module - 96 rxnsNew England BiolabsCatalog #E7595L
ReagentQubit® dsDNA HS Assay kitThermo Fisher ScientificCatalog #Q32854
ReagentNEB Blunt/TA Ligase Master Mix Catalog #M0367
ReagentAgencourt AMPure XP beadsBeckman CoulterCatalog #A63881
ReagentEthanolMerck Millipore (EMD Millipore)Catalog #100983
ReagentSuperScript™ IV First-Strand Synthesis SystemThermo FisherCatalog #18091200
ReagentONT MinION Flow Cell R9.4.1Oxford Nanopore TechnologiesCatalog #FLO-MIN106D
ReagentLigation sequencing kit 1DOxford Nanopore TechnologiesCatalog #SQK-LSK109
ReagentFlowcell Wash KitOxford Nanopore TechnologiesCatalog #EXP-WSH003
ReagentNEB Q5® Hot Start High-Fidelity 2X Master MixNew England BiolabsCatalog #M0494L
ReagentNative Barcoding Expansion 1-12 (PCR-free)Oxford Nanopore TechnologiesCatalog #EXP-NBD104
ReagentNative Barcoding Expansion 13-24 (PCR-free)Oxford Nanopore TechnologiesCatalog #EXP-NBD114
ReagentPrimer set SARS-CoV-2_v1
RT (Optional)
  • SuperScript™ IV First-Strand Synthesis System. (200 reactions) Cat: 18091200 Invitrogen

PCR (Optional)
  • Q5® Hot Start High-Fidelity 2X Master Mix Cat: M0494
  • Primer set SARS-CoV-2_v1 Pool 1 and Pool 2 (2kb) Paola Resende
Clean-up and QC
  • Qubit ds DNA HS kit. Cat: Q32854 Thermofisher
  • Ampure XP. Cat: A63881 Beckman Coulter

End-Prep, Barcoding, Adapter Ligation and Sequencing
  • NEBNext® Ultra™ II Ligation Module. Cat: E7595L NEB
  • NEBNext® Ultra™ II End Repair/dA-Tailing Module. Cat: E7546L NEB
  • NEB Blunt/TA Ligase Master Mix. Cat: M0287L NEB
  • Ligation Sequencing Kit. Cat: SQK-LSK109 ONT
  • Native Barcoding Expansion 1-12 (PCR-free). Cat: EXP-NBD104 ONT
  • Native Barcoding Expansion 13-24 (PCR-free). Cat: EXP-NBD114 ONT
  • Flow Cell (R9.4.1) ONT

Flow cell wash
  • Flow Cell Wash Kit. Cat: EXP-WSH003 ONT

Real-time data analysis
Primers dilution
Primers dilution
Instructions for Primer Scheme dilution:
Note
The primer scheme dilution should be prepared in Master Mix Hood or Clean Room.
To avoid cross-contamination make sure that your original stock reagents have no contact with RNA or any amplified DNA material.

If lyophilised each primer should be resuspended to Concentration100 micromolar (µM) (Stock dilution 1)

The primers from Primer Scheme 2kb_v1 Pool 1 and Primer Scheme 2kb_v1 Pool 2 are:

Primer Scheme 2kb_v1 Pool 1: F1, R1, F3, R3, F5, R5, F7, R7, F9, R9, F11, R11, F13, R13, F15, R15, F17, R17
(9 primers pairs)

Primer Scheme 2kb_v1 Pool 2: F2, R2, F4, R4, F6, R6, F8, R8, F10, R10, F12, R12, F14, R14, F16, R16
(8 primers pairs)

Primer Scheme 2kb_v1 Pool 1Primer Scheme 2kb_v1 Pool 2
Oligo nameOligo sequence (5' to 3')Oligo nameOligo sequence (5' to 3')
hCoV_F1_2kbACCAACCAACTTTCGATCTCTTGThCoV_F2_2kbCTGCTCAAAATTCTGTGCGTGT
hCoV_R1_2kbACACCACCTGTAATGTAGGCCAhCoV_R2_2kbGGTCAGCACCAAAAATACCAGCT
hCoV_F3_2kbAGCGGACACAATCTTGCTAAACAhCoV_F4_2kbTTGTGCACTTATCTTAGCCTACTGT
hCoV_R3_2kbGGTTGTCTGCTGTTGTCCACAAhCoV_R4_2kbTGCCAAAAACCACTCTGCAACT
hCoV_F5_2kbCACTATTGCAACCTACTGTACTGGThCoV_F6_2kbGTACACTGACTTTGCAACATCAGC
hCoV_R5_2kbCGTGTGTCAGGGCGTAAACTTThCoV_R6_2kbAACGGCAATTCCAGTTTGAGCA
hCoV_F7_2kbTGTACGCTGCTGTTATAAATGGAGAhCoV_F8_2kbTGGTACAACATTTACTTATGCATCAGC
hCoV_R7_2kbTTTGACAGCAGAATTGGCCCTThCoV_R8_2kbTGGGTGGTATGTCTGATCCCAA
hCoV_F9_2kbCCTTGACCAGGGCTTTAACTGChCoV_F10_2kbAGCAAAATGTTGGACTGAGACTGA
hCoV_R9_2kbATCATCTACAAAACAGCCGGCChCoV_R10_2kbCCAAGCAGGGTTACGTGTAAGG
hCoV_F11_2kbGCTGAAATTGTTGACACTGTGAGThCoV_F12_2kbTGCATTCCACACACCAGCTTTT
hCoV_R11_2kbAGCACCACCTAAATTGCAACGThCoV_R12_2kbTAACAAAGGCTGTCCACCATGC
hCoV_F13_2kbACAAAAGAAAATGACTCTAAAGAGGGTTThCoV_F14_2kbCAGGCTGCGTTATAGCTTGGAA
hCoV_R13_2kbTGTGCTACCGGCCTGATAGATThCoV_R14_2kbCATGACAAATGGCAGGAGCAGT
hCoV_F15_2kbTCAGAGTGTGTACTTGGACAATCAAhCoV_F16_2kbACGTGAGTCTTGTAAAACCTTCTTTTT
hCoV_R15_2kbGTACCGTTGGAATCTGCCATGGhCoV_R16_2kbACTGCCAGTTGAATCTGAGGGT
hCoV_F17_2kbGGAATCATCACAACTGTAGCTGCA
hCoV_R17_2kbTAGGCAGCTCTCCCTAGCATTG
Primer scheme to recover 2 kilobases amplicon of SARS-CoV-2 genome.

Prepare the Primer Scheme 2kb_v1 Pool 1 and Pool 2 Concentration100 micromolar (µM) (stock dilution 2)

Add Amount20 µL of each primer Concentration100 micromolar (µM) (stock dilution 1) to a 1.5mL tube labelled as Primer Scheme 2kb_v1 Pool 1. The final volume will be Amount360 µL

Add Amount20 µL of each primer Concentration100 micromolar (µM) (stock dilution 1) to a 1.5mL tube labelled as Primer Scheme 2kb_v1 Pool 2. The final volume will be Amount320 µL
Prepare the Primer Scheme 2kb_v1 Pool 1 and Pool 2 Concentration10 micromolar (µM) (concentration to be used)

Add Amount20 µL of each primer Concentration100 micromolar (µM) of Primer Scheme 2kb_v1 Pool 1 (stock dilution 2) to a 1.5mL tube labelled as Primer Scheme 2kb_v1 Pool 1 Concentration10 micromolar (µM)
Add Amount180 µL of water nuclease-free. The final volume will be Amount200 µL

Add Amount20 µL of each primer Concentration100 micromolar (µM) of Primer Scheme 2kb_v1 Pool 2 (stock dilution 2) to a 1.5mL tube labelled as Primer Scheme 2kb_v1 Pool 2 Concentration10 micromolar (µM)
Add Amount180 µL of water nuclease-free. The final volume will be Amount200 µL

Master Mix for cDNA and PCR steps
Master Mix for cDNA and PCR steps
30m
30m
Instructions to prepare the Maxter Mix for cDNA and PCR steps
Note
You can save time if you prepare both Master Mix cDNA and PCR to be used on the same day.

Note
The master mix for cDNA and PCR step should be prepared in Master Mix Hood or Clean Room. To avoid cross-contamination make sure that your original stock reagents have no contact with RNA or any amplified DNA material.

Note
A Negative Control (H2O nuclease free) should be included from cDNA step until the end.

Note
Keep the enzymes on ice and thaw the other reagents at room temperature before placing on ice.

Master Mix RT_1:

Note
This master Mix is prepared with components from:

SuperScript™ IV First-Strand Synthesis System (Catalog number: 18091050 [50 reactions] or 18091200 [200 reactions] ).

However, you can use separated items using the enzyme: SuperScript™ IV Reverse Transcriptase (Catalog number: 18090200).


Prepare the following components in a 0.2mL 8-strip tube for the number of samples that will be tested (positive samples + a negative control)

VOLUME COMPONENT

Amount1 µL 50µM random hexamers
Amount1 µL 10mM dNTPs mix (10mM each)
Master Mix RT_2:

Prepare the following components in a 1.5mL tube and keep the Master Mix2-RT on ice. (7uL per sample)
VOLUME COMPONENT

Amount4 µL 5x SSIV Buffer
Amount1 µL 100mM DTT
Amount1 µL RNAseOUT RNase Inhibitor
Amount1 µL SSIV Reverse transcriptase
Master Mix PCR Pool 1 and Pool 2:


Note
We have tested two catalog numbers of Q5®Hot Start High-Fidelity enzyme options:
Option 1 - Q5®Hot Start High-Fidelity 2X Master Mix (Catalog number: M0494)*
*Advantage: the master mix is ready to be used.

Option 2 - Q5®Hot Start High-Fidelity DNA Polymerase (Catalog number: M0493)

Bellow, we can find the master mix recipe of both. Please choose just one to continue the protocol.




Option 1 - Q5®Hot Start High-Fidelity 2X Master Mix (Catalog number: M0494)

Prepare the following components in two 1.5mL tubes and keep the Master Mix PCR Pool 1 and Pool 2 on ice

VOLUME VOLUME COMPONENT
Poo1 Pool 2

Amount12.5 µL Amount12.5 µL Q5 High-Fidelity 2X Master Mix
Amount3.6 µL Amount3.6 µL Primer Pool 1 or Primer Pool 2 (Concentration10 micromolar (µM) )
Amount6.4 µL Amount6.4 µL H2O Nuclease free


Mix the master mix by inversion several times, briefly spin to collect the contents at the bottom of the tube.
Dispense Amount22.5 µL per tube, 0.2mL 8-strip PCR tubes Pool 1 and 0.2mL 8-strip PCR tubes Pool 2.



Option 2 - Q5®Hot Start High-Fidelity DNA Polymerase (Catalog number: M0493)

VOLUME VOLUME COMPONENT
Poo1 Pool 2

Amount5 µL Amount5 µL 5X Q5 Reaction Buffer
Amount0.5 µL Amount0.5 µL 10 mM dNTPs
Amount0.25 µL Amount0.25 µL Q5 Hot Start DNA Polymerase
Amount3.6 µL Amount3.6 µL Primer Pool 1 or Primer Pool 2 (Concentration10 micromolar (µM) )
Amount13.15 µL Amount13.15 µL H2O Nuclease free


Mix the master mix by inversion several times, briefly spin to collect the contents at the bottom of the tube.
Dispense Amount22.5 µL per tube, 0.2mL 8-strip PCR tubes Pool 1 and 0.2mL 8-strip PCR tubes Pool 2.


Note
Label the side of the tubes, not the lids as the marker may be removed by the heat in the PCR machine.



cDNA
cDNA
1h 30m
1h 30m
Instructions for the cDNA step:
Note
This step should be conducted in the pre PCR area.

Note
Keep all the Master Mix (cDNA_2 and PCR) in the fridge.

Set up the thermocycler for the following condition:

Temperature65 °C

Spin down the Master Mix RT_1;
Add Amount11 µL RNA to each 0.2 mL tube containing the Master Mix RT_1;
Mix by pipetting; and pulse centrifuge the tubes to collect the contents at the bottom of the tube.
Incubate the reaction for Temperature65 °C Duration00:05:00


5m
Once this step is completed add Amount7 µL of Master Mix RT_2 in each tube
Mix by pipetting and pulse centrifuge the tubes to collect the contents at the bottom of the tube.
Incubate the reaction in the thermocycler for:
Temperature42 °C Duration00:50:00
Temperature70 °C Duration00:10:00
Temperature4 °C hold

Note
The cDNA can be stored in Temperature-20 °C . If needed, it is a safe stop point.


Note
The remaining RNA should be stored in Temperature-80 °C


1h
PCR
PCR
Instructions for the PCR step:
Note
This step should be conducted in the pre PCR area.

Set up the thermocycler for the following conditions:


1 cycle
Temperature98 °C Duration00:02:00 Heat Activation
35 cycles
Temperature98 °C Duration00:00:10 Denaturation
Temperature65 °C Duration00:05:00 Annealing
1 cycle
Temperature4 °C Hold
Add Amount2.5 µL cDNA of each sample to each 0.2mL 8-strip PCR tube containing the Master Mix PCR Pool 1 and Master Mix PCR Pool 2

Mix by pipetting and pulse centrifuge the tubes to collect the contents at the bottom of the tube.
Incubate in the thermocycler following the conditions described in substep 4.1
Clean-up
Clean-up
Instructions for the DNA purification using magnetic beads AMPURE


Note
This step should be conducted in the PCR room

Note
Keep the Ampure beads and Qubit standards in room temperature Duration00:15:00 before start.

Note
Prepare FRESH Concentration80 % (v/v) ethanol. Do not use Concentration80 % (v/v) Ethanol prepared in the previous day.

After the RT-PCR, Pool 1 and Pool 2 can be mixed (final volume Amount50 µL )

Add an equal volume of AmpureXP PCR Clean-up beads (Amount50 µL ) to the tube (ratio of 1:1 of Ampure beads).

Mix gently by either flicking or pipetting 8-10 times.
Note
If long reads (2 Kb) avoid the vortex

Incubate for Duration00:05:00 at room temperature.

Pulse centrifuge the tubes to remove any beads or solution from the lid or side of the tube.

Place on a magnetic rack and incubate for Duration00:02:00 or until the beads have pelleted against the magnet and the solution is completely clear.

Carefully remove and discard the solution, being careful not to displace the bead pellet.
Add Amount200 µL of fresh room-temperature Concentration80 % (v/v) Ethanol to the pellet.

Incubate for Duration00:01:00 .

Carefully remove and discard Ethanol, being careful not to displace the bead pellet.
Repeat Ethanol wash steps  13-15  to wash the pellet again and continue from step 17.
Briefly pulse centrifuge the pellet and carefully remove as much ethanol as possible using a Amount10 µL tip.

Allow the pellet to dry for Duration00:02:00 , being careful not to over-dry (if the pellet is cracking, then it is too dry). Pellet should appear opaque and slightly shiny.

Resuspend the pellet thoroughly in Amount32 µL of water, and incubate for Duration00:02:00

Pulse centrifuge to remove content in the lid.
Place on magnet and CAREFULLY remove water and transfer Amount32 µL to a clean 1.5 mL Eppendorf tube.


Note
MAKE SURE that no beads are transferred into this tube. In some cases, pulse centrifugation can be used to pellet residual beads.

Quality Control (QC)
Quality Control (QC)
Instructions to measure the amount of DNA
Note
Quantify Amount2 µL of the amplicon library using the Qubit fluorometer following the dsDNA protocol.


Note
To avoid any cross-contamination, we recommend that you remove the total amount of working solution required for your samples and standards from the working solution bottle and then add the required volume to the appropriate tubes instead of pipetting directly from the bottle to each tube.

Set up the required number of 0.5mL tubes for standards and samples. The Qubit™ 1X dsDNA HS Assay requires 2 standards.
Label the tube lids. Do not label the side of the tube as this could interfere with the sample read. Label the lid of each standard tube correctly. Calibration of the Qubit™ Fluorometer requires the standards to be inserted into the instrument in the right order.
Add the Qubit™ 1X dsDNA 1X buffer to each tube standard tube (Amount190 µL ) and each sample tube (Amount198 µL )

Add Amount10 µL of each Qubit™ standards 1 and 2 to the appropriate tube.

Add Amount2 µL of each sample to the appropriate tube

Vortex all tubes and incubate at room temperature for Duration00:02:00 , then proceed to “Read standards and samples”.

Carefully record all results to perform the DNA normalisation to prepare the library.
Normalisation
Normalisation
30m
30m
Instructions to normalise the DNA to prepare the library

For the barcoding step is needed Amount12.5 µL of DNA in a concentration of 60ng (long reads) per sample.
Note
For an efficient barcoding step, we observe that DNA in the barcoding step should range of 25 to 50 fmol (2kb). Then, we are using 60ng per sample.

Note
This is a safe stop point.

End-prep and barcoding Master Mix
End-prep and barcoding Master Mix
Instructions to prepare the End-prep and barcoding Master Mix
Note
The End-prep and barcoding Master Mix should be prepared in Master Mix Hood or Clean Room. To avoid cross-contamination make sure that your original stock reagents have no contact with RNA or any amplified DNA material.

Master Mix End-prep:

Prepare the following components in a 1.5mL tube and keep the Master Mix PCR on ice.
VOLUME COMPONENT
Amount1.75 µL Ultra II End Prep Reaction Buffer
Amount0.75 µL Ultra II End Prep Enzyme Mix
Master Mix barcoding:

Prepare the following components in a 1.5mL tube and keep the Master Mix PCR on ice.
VOLUME COMPONENT
Amount17.5 µL Ultra II Ligation Master Mix
Amount0.5 µL Ligation Enhancer
End-prep
End-prep
Instructions to End-prep reaction
Note
This is a ‘one-pot ligation’ protocol for native barcoded ligation libraries.

Note
This step should be conducted in the PCR room

Add Amount2.5 µL of Master Mix End-prep to each tube containing Amount12.5 µL of pre-normalised DNA (~ 60ng for 2 kb) and mix well by pipetting.

Incubate in a thermocycler at Temperature20 °C for Duration00:10:00 then Temperature65 °C for Duration00:05:00

Place on ice for Duration00:00:30


Barcoding
Barcoding
25m
25m

Instructions to Barcoding reaction
Note
Use a SINGLE native barcode (NBXX) per biological sample.

Note
This step should be conducted in the PCR room


Add Amount2.5 µL NBXX barcode to each tube containing Amount15 µL DNA end-prepped.

Add Amount18 µL of the barcoding mix to each tube and mix well by pipetting. The total volume will be Amount35.5 µL .

Incubate:
Temperature20 °C Duration00:15:00
Temperature70 °C Duration00:10:00
Then place on ice.
Note
The Temperature70 °C incubation is to inactivate the DNA ligase to prevent barcode cross-ligation when reactions are pooled in the next step



25m
Pooling and clean-up
Pooling and clean-up
25m
25m
Instructions to pool all samples and perform a clean-up

Pool all barcoded samples together into a clean 1.5 ml Eppendorf tube. Amount35 µL per sample

Note
If the number of samples processed is more than 20 samples this pooling needs to be done in a 2.0mL Eppendorf tube.


Add a proportion 1:1 of AMPURE beads in the barcoded DNA pooled.
If necessary measure the volume of the above reaction and add the same volume of AMPURE beads, to get a 1:1 solution.
Incubate for Duration00:05:00 in the HulaMixer.

Spin down the liquid and place on a magnet rack for Duration00:03:00 or until clear.

Remove all the solutions without touch in the pellet
Add Amount500 µL of Concentration80 % (v/v) ethanol to the tube still on the magnetic rack to wash.

Remove the tube from the magnet rack and remove and turn 180º and place the tube again in the magnetic rack.
Remove and discard Concentration80 % (v/v) ethanol without disturbing the pellet.

Repeat steps 45 and 47 and after continue from step 49.
Spin down and remove residual Concentration80 % (v/v) ethanol and air dry for Duration00:02:00 .

2m
Resuspend in Amount32 µL EB.

Incubate off the magnetic rack for Duration00:02:00 and after spin down.

2m
Replace the magnetic rack for Duration00:02:00

Wait until clear, then carefully remove the solution and transfer to a clean 1.5 mL Eppendorf tube labeled such as the name of your processing file.


QC
QC
25m
25m
Remove Amount2 µL and assess concentration by Qubit as described in the previous section. Keep the records in your processing file.

Normalisation
Normalisation
25m
25m
Dilute 100-200 fmol (~ 150ng for 2kb) pooled sample to Amount30 µL in Elution Buffer (EB).

Adapter ligation
Adapter ligation
25m
25m
Set up the following adapter ligation reaction in a 1.5mL tube:

VOLUME COMPONENT

Amount30 µL Cleaned-up barcoded amplicon pools (100-200 fmol)
Amount10 µL NEBNext Quick Ligation Reaction Buffer (5X)
Amount5 µL AMII Adapter mix II
Amount5 µL Quick T4 DNA Ligase
Incubate at RT for Duration00:30:00

Note
Remove the SFB from the freezer and let equilibrate to room temperature to be used in the next step

Clean-up
Clean-up
25m
25m

Clean-up

After Duration00:30:00 incubation add Amount50 µL Ampure beads (1x the volume of the total reaction volume)

Amount30 µL Incubate for Duration00:05:00

Spin down.
Place on a magnetic rack until clear.
Remove supernatant.
Add Amount200 µL SFB. Remove and turn the tube in the magnetic rack.

Safety information
CAUTION: DO NOT USE Concentration80 % (v/v) ETHANOL


Place on a magnetic rack until clear.
Remove supernatant.
Repeat SFB wash
Spin down and remove residual SFB .

Note
SFB is used for short fragments < 3kb.
LFB is used just for long fragments > 3Kb.

Add Amount14 µL EB and resuspend by flicking.

Incubate at RT for Duration00:02:00

Place on a magnetic rack.
Carefully transfer the solution to a clean 1.5 mL Eppendorf tube.
QC
QC
25m
25m
Remove Amount2 µL and assess concentration by Qubit fluorometer- recovery aim 50-100 fmol, 61.80 - 123.6 ng for 2Kb amplicons


Note
The library can be now stored at 4°C overnight if required, but for best results, proceed immediately to sequencing.


Testing the flow cell
Testing the flow cell
25m
25m



Note
During this testing to save time you can thaw the following reagents at room temperature before placing on ice: Sequencing buffer (SQB); Loading beads (LB); Flush buffer (FB); and Flush tether (FLT). They will be used in the next step

Note
Choose a flow cell, record the name of the flow cell on your working sheet

Insert the flow cell in the MinION device or GridION.
Test the flow cell using the MinKnow software to observe the number of active pores, keep this record on your working sheet.
Select the flowcell ticking the box
Click “check flowcell”
Click “start test”.
Loading the flow cell
Loading the flow cell
25m
25m
For this step please follow the protocol described by Josh Quick below:
CITATION
Josh Quick. Priming and loading a MinION flowcell.
LINK
dx.doi.org/10.17504/protocols.io.7q5hmy6


MinKnow software
MinKnow software
25m
25m
Double–click the MinKNOW icon located on the desktop to open the MinKNOW software.
If your MinION was disconnected from the computer, plug it back in.
Choose the following flow cell type from the selector box: FLO-MIN106 : R9.4.1 flow cell
Then mark the flow cell as Selected.
Click the New Experiment button at the bottom left of the screen.
On the new experiment popup screen select the running parameters for your experiment from the individual tabs:

Experiment

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: Select LSK109. Do not select barcoding kits.

Run Options: Set the run length to 12 hours (you can stop the run once sufficient data has been collected as determined using RAMPART software).

Basecalling: Leave base calling turned but select 'high accuracy basecalling'.

Click 'Start run'.
Wash a flow cell
Wash a flow cell
40m
40m
Washing the flow cell:
Note
This step should be performed to reuse the flow cell after a previous run.

Note
The flow cell should be washed on the same day or on the following day of the run. Do not wait too much time to wash the Flow Cell.

For this step please follow the protocol described by Kirstyn Brunker bellow:
CITATION
Kirstyn Brunker. Washing a MinION flowcell.
LINK
dx.doi.org/10.17504/protocols.io.bddzi276

Citations
Quick J, Grubaugh ND, Pullan ST, Claro IM, Smith AD, Gangavarapu K, Oliveira G, Robles-Sikisaka R, Rogers TF, Beutler NA, Burton DR, Lewis-Ximenez LL, de Jesus JG, Giovanetti M, Hill SC, Black A, Bedford T, Carroll MW, Nunes M, Alcantara LC Jr, Sabino EC, Baylis SA, Faria NR, Loose M, Simpson JT, Pybus OG, Andersen KG, Loman NJ. Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples.
https://doi.org/10.1038/nprot.2017.066
Step 79
Josh Quick. Priming and loading a MinION flowcell
dx.doi.org/10.17504/protocols.io.7q5hmy6
Step 86
Kirstyn Brunker. Washing a MinION flowcell
dx.doi.org/10.17504/protocols.io.bddzi276