TITLE: Long reads nanopore sequencing to recover SARS-CoV-2 whole genome 
AUTHORS: Paola Resende

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">This protocol describes step-by-step instructions for building long (~2kb) amplicon libraries to recover SARS-CoV-2 genomes using the nanopore sequencing.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">It can be applied for sequencing on the MinION device or GridION. </div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">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. </div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">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. </div></div><div class = "text-block"><div class = "justify" style = "text-align:left">Manager: Rachel Williams    </div></div><div class = "text-block"><a href="https://www.ucl.ac.uk/infection-immunity/pathogen-genomics-unit" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.ucl.ac.uk/infection-immunity/pathogen-genomics-unit</span></a></div><div class = "text-block">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.</div><div class = "text-block">If you have doubts, we are happy to help you to test or implement the protocol.</div></div>

[STEPS]
?. [Primers dilution]
Instructions for Primer Scheme dilution:If lyophilised each primer should be resuspended to   (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)  ABCD1Primer Scheme 2kb_v1 Pool 1Primer Scheme 2kb_v1 Pool 22Oligo nameOligo sequence (5' to 3')Oligo nameOligo sequence (5' to 3')3hCoV_F1_2kbACCAACCAACTTTCGATCTCTTGThCoV_F2_2kbCTGCTCAAAATTCTGTGCGTGT4hCoV_R1_2kbACACCACCTGTAATGTAGGCCAhCoV_R2_2kbGGTCAGCACCAAAAATACCAGCT5hCoV_F3_2kbAGCGGACACAATCTTGCTAAACAhCoV_F4_2kbTTGTGCACTTATCTTAGCCTACTGT6hCoV_R3_2kbGGTTGTCTGCTGTTGTCCACAAhCoV_R4_2kbTGCCAAAAACCACTCTGCAACT7hCoV_F5_2kbCACTATTGCAACCTACTGTACTGGThCoV_F6_2kbGTACACTGACTTTGCAACATCAGC8hCoV_R5_2kbCGTGTGTCAGGGCGTAAACTTThCoV_R6_2kbAACGGCAATTCCAGTTTGAGCA9hCoV_F7_2kbTGTACGCTGCTGTTATAAATGGAGAhCoV_F8_2kbTGGTACAACATTTACTTATGCATCAGC10hCoV_R7_2kbTTTGACAGCAGAATTGGCCCTThCoV_R8_2kbTGGGTGGTATGTCTGATCCCAA11hCoV_F9_2kbCCTTGACCAGGGCTTTAACTGChCoV_F10_2kbAGCAAAATGTTGGACTGAGACTGA12hCoV_R9_2kbATCATCTACAAAACAGCCGGCChCoV_R10_2kbCCAAGCAGGGTTACGTGTAAGG13hCoV_F11_2kbGCTGAAATTGTTGACACTGTGAGThCoV_F12_2kbTGCATTCCACACACCAGCTTTT14hCoV_R11_2kbAGCACCACCTAAATTGCAACGThCoV_R12_2kbTAACAAAGGCTGTCCACCATGC15hCoV_F13_2kbACAAAAGAAAATGACTCTAAAGAGGGTTThCoV_F14_2kbCAGGCTGCGTTATAGCTTGGAA16hCoV_R13_2kbTGTGCTACCGGCCTGATAGATThCoV_R14_2kbCATGACAAATGGCAGGAGCAGT17hCoV_F15_2kbTCAGAGTGTGTACTTGGACAATCAAhCoV_F16_2kbACGTGAGTCTTGTAAAACCTTCTTTTT18hCoV_R15_2kbGTACCGTTGGAATCTGCCATGGhCoV_R16_2kbACTGCCAGTTGAATCTGAGGGT19hCoV_F17_2kbGGAATCATCACAACTGTAGCTGCA  20hCoV_R17_2kbTAGGCAGCTCTCCCTAGCATTG
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.
ABCD1Primer Scheme 2kb_v1 Pool 1Primer Scheme 2kb_v1 Pool 22Oligo nameOligo sequence (5' to 3')Oligo nameOligo sequence (5' to 3')3hCoV_F1_2kbACCAACCAACTTTCGATCTCTTGThCoV_F2_2kbCTGCTCAAAATTCTGTGCGTGT4hCoV_R1_2kbACACCACCTGTAATGTAGGCCAhCoV_R2_2kbGGTCAGCACCAAAAATACCAGCT5hCoV_F3_2kbAGCGGACACAATCTTGCTAAACAhCoV_F4_2kbTTGTGCACTTATCTTAGCCTACTGT6hCoV_R3_2kbGGTTGTCTGCTGTTGTCCACAAhCoV_R4_2kbTGCCAAAAACCACTCTGCAACT7hCoV_F5_2kbCACTATTGCAACCTACTGTACTGGThCoV_F6_2kbGTACACTGACTTTGCAACATCAGC8hCoV_R5_2kbCGTGTGTCAGGGCGTAAACTTThCoV_R6_2kbAACGGCAATTCCAGTTTGAGCA9hCoV_F7_2kbTGTACGCTGCTGTTATAAATGGAGAhCoV_F8_2kbTGGTACAACATTTACTTATGCATCAGC10hCoV_R7_2kbTTTGACAGCAGAATTGGCCCTThCoV_R8_2kbTGGGTGGTATGTCTGATCCCAA11hCoV_F9_2kbCCTTGACCAGGGCTTTAACTGChCoV_F10_2kbAGCAAAATGTTGGACTGAGACTGA12hCoV_R9_2kbATCATCTACAAAACAGCCGGCChCoV_R10_2kbCCAAGCAGGGTTACGTGTAAGG13hCoV_F11_2kbGCTGAAATTGTTGACACTGTGAGThCoV_F12_2kbTGCATTCCACACACCAGCTTTT14hCoV_R11_2kbAGCACCACCTAAATTGCAACGThCoV_R12_2kbTAACAAAGGCTGTCCACCATGC15hCoV_F13_2kbACAAAAGAAAATGACTCTAAAGAGGGTTThCoV_F14_2kbCAGGCTGCGTTATAGCTTGGAA16hCoV_R13_2kbTGTGCTACCGGCCTGATAGATThCoV_R14_2kbCATGACAAATGGCAGGAGCAGT17hCoV_F15_2kbTCAGAGTGTGTACTTGGACAATCAAhCoV_F16_2kbACGTGAGTCTTGTAAAACCTTCTTTTT18hCoV_R15_2kbGTACCGTTGGAATCTGCCATGGhCoV_R16_2kbACTGCCAGTTGAATCTGAGGGT19hCoV_F17_2kbGGAATCATCACAACTGTAGCTGCA  20hCoV_R17_2kbTAGGCAGCTCTCCCTAGCATTG
?. [Primers dilution]
Prepare the Primer Scheme 2kb_v1 Pool 1 and Pool 2  (stock dilution 2)Add  of each primer (stock dilution 1) to a 1.5mL tube labelled as Primer Scheme 2kb_v1 Pool 1. The final volume will be Add  of each primer (stock dilution 1) to a 1.5mL tube labelled as Primer Scheme 2kb_v1 Pool 2. The final volume will be
20 µl
360 µl
20 µl
320 µl
?. [Primers dilution]
Prepare the Primer Scheme 2kb_v1 Pool 1 and Pool 2 (concentration to be used)Add  of each primer  of Primer Scheme 2kb_v1 Pool 1 (stock dilution 2) to a 1.5mL tube labelled as Primer Scheme 2kb_v1 Pool 1  Add   of water nuclease-free. The final volume will be Add  of each primer  of Primer Scheme 2kb_v1 Pool 2 (stock dilution 2) to a 1.5mL tube labelled as Primer Scheme 2kb_v1 Pool 2 Add  of water nuclease-free. The final volume will be
20 µl
180 µl
200 µl
20 µl
180 µl
200 µl
?. [Master Mix for cDNA and PCR steps]
Instructions to prepare the Maxter Mix for cDNA and PCR steps
You can save time if you prepare both Master Mix cDNA and PCR to be used on the same day.
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.
A Negative Control (H2O nuclease free) should be included from cDNA step until the end.
Keep the enzymes on ice and thaw the other reagents at room temperature before placing on ice.
?. [Master Mix for cDNA and PCR steps]
Master Mix RT_1: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         50µM random hexamers         10mM dNTPs mix (10mM each)
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).
1 µl
1 µl
?. [Master Mix for cDNA and PCR steps]
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        5x SSIV Buffer        100mM DTT        RNAseOUT RNase Inhibitor        SSIV Reverse transcriptase
4 µl
1 µl
1 µl
1 µl
?. [Master Mix for cDNA and PCR steps]
Master Mix PCR Pool 1 and Pool 2: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 iceVOLUME              VOLUME                  COMPONENT   Poo1                    Pool 2                                              Q5 High-Fidelity 2X Master Mix              Primer Pool 1 or Primer Pool 2 ()              H2O Nuclease free  Mix the master mix by inversion several times, briefly spin to collect the contents at the bottom of the tube.Dispense  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                                                5X Q5 Reaction Buffer          10 mM dNTPs       Q5 Hot Start DNA Polymerase          Primer Pool 1 or Primer Pool 2 ()     H2O Nuclease freeMix the master mix by inversion several times, briefly spin to collect the contents at the bottom of the tube.Dispense  per tube, 0.2mL 8-strip PCR tubes Pool 1 and 0.2mL 8-strip PCR tubes Pool 2.
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.
12.5 µl
12.5 µl
3.6 µl
3.6 µl
6.4 µl
6.4 µl
22.5 µl
5 µl
5 µl
0.5 µl
0.5 µl
0.25 µl
0.25 µl
3.6 µl
3.6 µl
13.15 µl
13.15 µl
22.5 µl
Label the side of the tubes, not the lids as the marker may be removed by the heat in the PCR machine.
?. [cDNA]
Instructions for the cDNA step:
This step should be conducted in the pre PCR area.
Keep all the Master Mix (cDNA_2 and PCR) in the fridge.
?. [cDNA]
Set up the thermocycler for the following condition:
65 °C
?. [cDNA]
Spin down the Master Mix RT_1;
?. [cDNA]
Add   RNA to each 0.2 mL tube containing the Master Mix RT_1;
11 µl
?. [cDNA]
Mix by pipetting; and pulse centrifuge the tubes to collect the contents at the bottom of the tube.
?. [cDNA]
Incubate the reaction for
65 °C
?. [cDNA]
Once this step is completed add  of Master Mix RT_2 in each tube
7 µl
?. [cDNA]
Mix by pipetting and pulse centrifuge the tubes to collect the contents at the bottom of the tube.
?. [cDNA]
Incubate the reaction in the thermocycler for: hold
42 °C
70 °C
4 °C
The cDNA can be stored in   . If needed, it is a safe stop point.
The remaining RNA should be stored in
?. [PCR]
Instructions for the PCR step:
This step should be conducted in the pre PCR area.
?. [PCR]
Set up the thermocycler for the following conditions: 1 cycle     Heat Activation 35 cycles    Denaturation    Annealing1 cycle   Hold
98 °C
98 °C
65 °C
4 °C
?. [PCR]
Add  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
2.5 µl
?. [PCR]
Mix by pipetting and pulse centrifuge the tubes to collect the contents at the bottom of the tube.
?. [PCR]
Incubate in the thermocycler following the conditions described in substep 4.1
?. [Clean-up]
Instructions for the DNA purification using magnetic beads AMPURE
This step should be conducted in the PCR room
Keep the Ampure beads and Qubit standards in room temperature   before start.
Prepare FRESH   ethanol. Do not use   Ethanol prepared in the previous day.
?. [Clean-up]
After the RT-PCR, Pool 1 and Pool 2 can be mixed (final volume )
50 µl
?. [Clean-up]
Add an equal volume of AmpureXP PCR Clean-up beads () to the tube  (ratio of 1:1 of Ampure beads).
50 µl
?. [Clean-up]
Mix gently by either flicking or pipetting 8-10 times.
If long reads (2 Kb) avoid the vortex
?. [Clean-up]
Incubate for  at room temperature.
?. [Clean-up]
Pulse centrifuge the tubes to remove any beads or solution from the lid or side of the tube.
?. [Clean-up]
Place on a magnetic rack and incubate for  or until the beads have pelleted against the magnet and the solution is completely clear.
?. [Clean-up]
Carefully remove and discard the solution, being careful not to displace the bead pellet.
?. [Clean-up]
Add of fresh room-temperature  Ethanol to the pellet.
200 µl
?. [Clean-up]
Incubate for .
?. [Clean-up]
Carefully remove and discard Ethanol, being careful not to displace the bead pellet.
?. [Clean-up]
Repeat Ethanol wash steps  13-15  to wash the pellet again and continue from step 17.
?. [Clean-up]
Briefly pulse centrifuge the pellet and carefully remove as much ethanol as possible using a  tip.
10 µl
?. [Clean-up]
Allow the pellet to dry for , being careful not to over-dry (if the pellet is cracking, then it is too dry). Pellet should appear opaque and slightly shiny.
?. [Clean-up]
Resuspend the pellet thoroughly in of water, and incubate for
32 µl
?. [Clean-up]
Pulse centrifuge to remove content in the lid.
?. [Clean-up]
Place on magnet and CAREFULLY remove water and transfer to a clean 1.5 mL Eppendorf tube.
32 µl
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)]
Instructions to measure the amount of DNA
Quantify  of the amplicon library using the Qubit fluorometer following the dsDNA protocol.
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.
?. [Quality Control (QC)]
Set up the required number of 0.5mL tubes for standards and samples. The Qubit™ 1X dsDNA HS Assay requires 2 standards.
?. [Quality Control (QC)]
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.
?. [Quality Control (QC)]
Add the Qubit™ 1X dsDNA 1X buffer to each tube standard tube () and each sample tube ()
190 µl
198 µl
?. [Quality Control (QC)]
Add of each Qubit™ standards 1 and 2 to the appropriate tube.
10 µl
?. [Quality Control (QC)]
Add of each sample to the appropriate tube
2 µl
?. [Quality Control (QC)]
Vortex all tubes and incubate at room temperature for , then proceed to “Read standards and samples”.
?. [Quality Control (QC)]
Carefully record all results to perform the DNA normalisation to prepare the library.
?. [Normalisation]
Instructions to normalise the DNA to prepare the libraryFor the barcoding step is needed of DNA in a concentration of 60ng (long reads) per sample.
12.5 µl
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.
This is a safe stop point.
?. [End-prep and barcoding Master Mix]
Instructions to prepare the End-prep and barcoding Master Mix
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.
?. [End-prep and barcoding Master Mix]
Master Mix End-prep:Prepare the following components in a 1.5mL tube and keep the Master Mix PCR on ice. VOLUME        COMPONENT  Ultra II End Prep Reaction Buffer  Ultra II End Prep Enzyme Mix
1.75 µl
0.75 µl
?. [End-prep and barcoding Master Mix]
Master Mix barcoding:Prepare the following components in a 1.5mL tube and keep the Master Mix PCR on ice. VOLUME        COMPONENT  Ultra II Ligation Master Mix     Ligation Enhancer
17.5 µl
0.5 µl
?. [End-prep]
Instructions to End-prep reaction
This is a ‘one-pot ligation’ protocol for native barcoded ligation libraries.
This step should be conducted in the PCR room
?. [End-prep]
Add  of Master Mix End-prep to each tube containing  of pre-normalised DNA (~ 60ng for 2 kb) and mix well by pipetting.
2.5 µl
12.5 µl
?. [End-prep]
Incubate in a thermocycler at  for  then  for
20 °C
65 °C
?. [End-prep]
Place on ice for
?. [Barcoding]
Instructions to Barcoding reaction
Use a SINGLE native barcode (NBXX) per biological sample.
This step should be conducted in the PCR room
?. [Barcoding]
Add NBXX barcode to each tube containing DNA end-prepped.
2.5 µl
15 µl
?. [Barcoding]
Add of the barcoding mix to each tube and mix well by pipetting. The total volume will be .
18 µl
35.5 µl
?. [Barcoding]
Incubate:Then place on ice.
20 °C
70 °C
The   incubation is to inactivate the DNA ligase to prevent barcode cross-ligation when reactions are pooled in the next step
?. [Pooling and clean-up]
Instructions to pool all samples and perform a clean-upPool all barcoded samples together into a clean 1.5 ml Eppendorf tube.  per sample
35 µl
If the number of samples processed is more than 20 samples this pooling needs to be done in a 2.0mL Eppendorf tube.
?. [Pooling and clean-up]
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.
?. [Pooling and clean-up]
Incubate for  in the HulaMixer.
?. [Pooling and clean-up]
Spin down the liquid and place on a magnet rack for  or until clear.
?. [Pooling and clean-up]
Remove all the solutions without touch in the pellet
?. [Pooling and clean-up]
Add  of  ethanol to the tube still on the magnetic rack to wash.
500 µl
?. [Pooling and clean-up]
Remove the tube from the magnet rack and remove and turn 180º and place the tube again in the magnetic rack.
?. [Pooling and clean-up]
Remove and discard  ethanol without disturbing the pellet.
?. [Pooling and clean-up]
Repeat steps 45 and 47 and after continue from step 49.
?. [Pooling and clean-up]
Spin down and remove residual  ethanol and air dry for .
?. [Pooling and clean-up]
Resuspend in  EB.
32 µl
?. [Pooling and clean-up]
Incubate off the magnetic rack for   and after spin down.
?. [Pooling and clean-up]
Replace the magnetic rack for
?. [Pooling and clean-up]
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]
Remove  and assess concentration by Qubit as described in the previous section. Keep the records in your processing file.
2 µl
?. [Normalisation]
Dilute 100-200 fmol (~ 150ng for 2kb) pooled sample to  in Elution Buffer (EB).
30 µl
?. [Adapter ligation]
Set up the following adapter ligation reaction in a 1.5mL tube:VOLUME     COMPONENT   Cleaned-up barcoded amplicon pools (100-200 fmol)   NEBNext Quick Ligation Reaction Buffer (5X)      AMII Adapter mix II      Quick T4 DNA Ligase
30 µl
10 µl
5 µl
5 µl
?. [Adapter ligation]
Incubate at RT for
Remove the SFB from the freezer and let equilibrate to room temperature to be used in the next step
?. [Clean-up]
Clean-upAfter  incubation add  Ampure beads (1x the volume of the total reaction volume)
50 µl
?. [Clean-up]
Incubate for
30 µl
?. [Clean-up]
Spin down.
?. [Clean-up]
Place on a magnetic rack until clear.
?. [Clean-up]
Remove supernatant.
?. [Clean-up]
Add  SFB. Remove and turn the tube in the magnetic rack.
200 µl
CAUTION: DO NOT USE   ETHANOL
?. [Clean-up]
Place on a magnetic rack until clear.
?. [Clean-up]
Remove supernatant.
?. [Clean-up]
Repeat SFB wash
?. [Clean-up]
Spin down and remove residual SFB .
SFB is used for short fragments LFB is used just for long fragments > 3Kb.
?. [Clean-up]
Add EB and resuspend by flicking.
14 µl
?. [Clean-up]
Incubate at RT for
?. [Clean-up]
Place on a magnetic rack.
?. [Clean-up]
Carefully transfer the solution to a clean 1.5 mL Eppendorf tube.
?. [QC]
Remove and assess concentration by Qubit fluorometer- recovery aim 50-100 fmol, 61.80 - 123.6 ng for 2Kb amplicons
2 µl
The library can be now stored at 4°C overnight if required, but for best results, proceed immediately to sequencing.
?. [Testing the flow cell]
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
Choose a flow cell, record the name of the flow cell on your working sheet
?. [Testing the flow cell]
Insert the flow cell in the MinION device or GridION.
?. [Testing the flow cell]
Test the flow cell using the MinKnow software to observe the number of active pores, keep this record on your working sheet.
?. [Testing the flow cell]
Select the flowcell ticking the box
?. [Testing the flow cell]
Click “check flowcell”
?. [Testing the flow cell]
Click “start test”.
?. [Loading the flow cell]
For this step please follow the protocol described by Josh Quick below:
?. [MinKnow software]
Double–click the MinKNOW icon located on the desktop to open the MinKNOW software.
?. [MinKnow software]
If your MinION was disconnected from the computer, plug it back in.
?. [MinKnow software]
Choose the following flow cell type from the selector box:  FLO-MIN106 : R9.4.1 flow cell
?. [MinKnow software]
Then mark the flow cell as Selected.
?. [MinKnow software]
Click the New Experiment button at the bottom left of the screen.
?. [MinKnow software]
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 tabsExperiment: 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]
Washing the flow cell:For this step please follow the protocol described by Kirstyn Brunker bellow:
This step should be performed to reuse the flow cell after a previous run.
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.