TITLE: Nanopore genome sequencing with barcode
AUTHORS: phangguanjie

[DESCRIPTION]
The modified version of official protocol using Kappa chemicals

[STEPS]
SECTION: Step1. End-prep &amp; A-Tailing
1. In a 200μl PCR tube, mix the following:

 

SECTION: Step1. End-prep &amp; A-Tailing
1.1. Ensure the components are thoroughly mixed by pipetting, and spin down.
SECTION: Step1. End-prep &amp; A-Tailing
1.2. Using a thermal cycler, incubate at 20 °C for30 min and65 °C for 30 min. Hold at4 °C

SECTION: Step2. Barcode Ligation
2. Thaw LNB, Native Barcode at room temperature, spin down and mix by vortex.

 

SECTION: Step2. Barcode Ligation
2.1. In a 200μl PCR tube, mix in the following order:

 

SECTION: Step2. Barcode Ligation
2.2. Ensure the components are thoroughly mixed by pipetting, and spin down.
SECTION: Step2. Barcode Ligation
2.3. Using a thermal cycler, incubate at 20 °C30 min .

 

 

SECTION: Step3. Barcode Ligation Products clean-up
3. Add 44 ul (0.8X) of resuspended HyperPure beads to the barcode-ligated reaction and mix by flicking the tube.
SECTION: Step3. Barcode Ligation Products clean-up
3.1. Incubate on a Hula mixer (rotator mixer) for 5 minutes at RT.
SECTION: Step3. Barcode Ligation Products clean-up
3.2. Prepare 500 ul of fresh 70% ethanol in Nuclease-free water.
SECTION: Step3. Barcode Ligation Products clean-up
3.3. Spin down the sample and pellet on a magnetic stand.
SECTION: Step3. Barcode Ligation Products clean-up
3.4. Keep the tube on the magnetic stand, and pipette off the supernatant.
SECTION: Step3. Barcode Ligation Products clean-up
3.5. Keep the tube on the magnetic stand and wash the beads with 200 ul of freshly-prepared 70% ethanol without disturbing the pellet.
SECTION: Step3. Barcode Ligation Products clean-up
3.6. Remove the ethanol using a pipette and discard.
SECTION: Step3. Barcode Ligation Products clean-up
3.7. Repeat the previous step to remove ethanol.
SECTION: Step3. Barcode Ligation Products clean-up
3.8. Spin down and place the tube back on the magnetic stand.
SECTION: Step3. Barcode Ligation Products clean-up
3.9. Pipette off any residual ethanol.
SECTION: Step3. Barcode Ligation Products clean-up
3.10. Allow to dry for ~30 seconds(1-3 min), but do not dry the pellet to the point of cracking.
SECTION: Step3. Barcode Ligation Products clean-up
3.11. Remove the tube from the magnetic stand and resuspend the pellet in 11 ul Nuclease-free water.(55 °C 1 min)

SECTION: Step3. Barcode Ligation Products clean-up
3.12. Pellet the beads on a magnetic stand until the eluate is clear and colourless.
SECTION: Step3. Barcode Ligation Products clean-up
3.13. Remove and retain 11 ul of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
SECTION: Step3. Barcode Ligation Products clean-up
3.14. Quantify 1 μl of eluted sample using a Quantus fluorometer.


SECTION: Step4. Adapter Ligation (AMII)
4. Pool samples to be totaly 100 fmol DNA mix.
SECTION: Step4. Adapter Ligation (AMII)
4.1. 
 

SECTION: Step4. Adapter Ligation (AMII)
4.2. Using a thermal cycler, incubate at 20 °C30 min .
 


 

SECTION: Step5. DNA library clean-up
5. Add 44 ul (0.4x beads) of resuspended HyperPuer beads to the adapter-ligated reaction and mix by pipetting. 
SECTION: Step5. DNA library clean-up
5.1. Incubate on a Hula mixer (rotator mixer) for 5 minutes at RT.
SECTION: Step5. DNA library clean-up
5.2. Place on magnetic stand, allow beads to pellet and pipette off supernatant.
SECTION: Step5. DNA library clean-up
5.3. Wash the beads by adding either 250 μl Long Fragment Buffer (LFB) or 250 μl S Fragment Buffer (SFB)(<3k).
SECTION: Step5. DNA library clean-up
5.4. Flick the beads to resuspend, then return the tube to the magnetic stand and allow the beads to pellet.
SECTION: Step5. DNA library clean-up
5.5. Remove the supernatant using a pipette and discard.
SECTION: Step5. DNA library clean-up
5.6. Wash the beads by adding either 250 μl Long Fragment Buffer (LFB) or 250 μl S Fragment Buffer (SFB)(<3k).
SECTION: Step5. DNA library clean-up
5.7. Flick the beads to resuspend, then return the tube to the magnetic stand and allow the beads to pellet.
SECTION: Step5. DNA library clean-up
5.8. Remove the supernatant using a pipette and discard.
SECTION: Step5. DNA library clean-up
5.9. Spin down and place the tube back on the magnetic stand.
SECTION: Step5. DNA library clean-up
5.10. Pipette off any residual supernatant.
SECTION: Step5. DNA library clean-up
5.11. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
SECTION: Step5. DNA library clean-up
5.12. Remove the tube from the magnetic stand and resuspend the pellet in 14 ul Elution Buffer.
SECTION: Step5. DNA library clean-up
5.13. Keep at RT for 10 min.
SECTION: Step5. DNA library clean-up
5.14. Pellet the beads on a magnetic stand until the eluate is clear and colourless.
SECTION: Step5. DNA library clean-up
5.15. Remove and retain 14 ul of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
SECTION: Step5. DNA library clean-up
5.16. Quantify 1 μl of eluted sample using a Quantus fluorometer.
 

SECTION: Step6. Priming and loading
6. During incubation, take out SQB, FLT, FLB from the frige 30 minutes earlier to thaw on ice.
SECTION: Step6. Priming and loading
6.1. Thaw the flow cell, Sequencing Buffer (SQB), Loading Beads (LB), Flush Tether (FLT) and one tube of Flush Buffer (FB) at room temperature. 
SECTION: Step6. Priming and loading
6.2. Open the GridION and slide the flow cell under the clip.

 

SECTION: Step6. Priming and loading
6.3. Check flow cell (Check the pore)

 

SECTION: Step6. Priming and loading
6.4. Mix the Sequencing Buffer (SQB), Flush Tether (FLT) and Flush Buffer (FB) tubes by pipetting and spin down at room temperature.
SECTION: Step6. Priming and loading
6.5. Remove the flow cell from the machine and slide the priming port cover clockwise to open the priming port.
SECTION: Step6. Priming and loading
6.6. After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles (a few μl):
1. Set a P1000 pipette to 200 μl.
2. Insert the tip into the priming port.
3. Turn the wheel until the dial shows 220-230 μl, or until you can see a small volume of buffer entering the pipette tip.

 

SECTION: Step6. Priming and loading
6.7. To prepare the flow cell priming mix, add 30 μl of thawed and mixed Flush Tether (FLT) directly to the tube of thawed and mixed Flush Buffer (FB), and mix by vortexing at room temperature.

 

SECTION: Step6. Priming and loading
6.8. Load 800 μl of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait for 5 minutes. During this time, prepare the library for loading by following the steps below.
 

SECTION: Step6. Priming and loading
6.9. Thoroughly mix the contents of the Loading Beads (LB) by flick.
SECTION: Step6. Priming and loading
6.10. In a new tube, prepare the library for loading as follows:
 

SECTION: Step6. Priming and loading
6.11. Complete the flow cell priming:
1. Gently lift the SpotON sample port cover to make the SpotON sample port accessible.
2. Load 200 μl of the priming mix into the flow cell via the priming port (not the SpotON sample port), avoiding the introduction of air bubbles.
 

SECTION: Step6. Priming and loading
6.12. Mix the prepared library gently by pipetting up and down just prior to loading.
SECTION: Step6. Priming and loading
6.13. Add 75 μl of sample to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next.
SECTION: Step6. Priming and loading
6.14. Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the priming port and replace the GridION.
SECTION: Step7.Flow Cell Wash
7. Place the tube of Wash Mix (WMX) on ice. Do not vortex the tube.
SECTION: Step7.Flow Cell Wash
7.1. Thaw one tube of Wash Diluent (DIL) at room temperature.
SECTION: Step7.Flow Cell Wash
7.2. Mix the contents of Wash Diluent (DIL) thoroughly by vortexing, spin down briefly and place on ice.
SECTION: Step7.Flow Cell Wash
7.3. In a clean 1.5 ml Eppendorf DNA tube, prepare the following Flow Cell Wash Mix:
SECTION: Step7.Flow Cell Wash
7.4. 
 

SECTION: Step7.Flow Cell Wash
7.5. Mix well by pipetting, and place on ice. Do not vortex the tube.
SECTION: Step7.Flow Cell Wash
7.6. Stop or pause the sequencing experiment in MinKNOW, and leave the flow cell in the device.
SECTION: Step7.Flow Cell Wash
7.7. Ensure that the priming port cover and SpotON sample port cover are in the positions indicated in the figure below.
SECTION: Step7.Flow Cell Wash
7.8. Using a P1000, remove all fluid from the waste channel through Waste port 1. 
As both the priming port and SpotON sample port are closed, no fluid should leave the sensor array area.
SECTION: Step7.Flow Cell Wash
7.9. Rotate the flow cell priming port cover clockwise so that the priming port is visible.
SECTION: Step7.Flow Cell Wash
7.10. Check for air between the priming port and the sensor array. If necessary, using a P1000 draw back a small volume to remove any air (a few μls):
1. Set a P1000 pipette to 200 μl
2. Insert the tip into the priming port
3. Turn the wheel until the dial shows 220-230 μl, or until you can see a small volume of buffer/liquid entering the pipette tip.
4. Visually check that there is continuous buffer from the priming port across the sensor array.
SECTION: Step7.Flow Cell Wash
7.11. Load 400 μl of the prepared Flow Cell Wash Mix into the flow cell via the priming port, avoiding the introduction of air.
SECTION: Step7.Flow Cell Wash
7.12. Close the priming port and wait for 30 minutes.
SECTION: Step7.Flow Cell Wash
7.13. Ensure that the priming port cover and SpotON sample port cover are in the positions indicated in the figure below.
SECTION: Step7.Flow Cell Wash
7.14. Using a P1000, remove all fluid from the waste channel through Waste port 1. As both the priming port and SpotON sample port are closed, no fluid should leave the sensor array area.

 

SECTION: Step8. Store the MinION/GridION flow cell for later use
8. Thaw one tube of Storage Buffer (S) at room temperature.
SECTION: Step8. Store the MinION/GridION flow cell for later use
8.1. Mix contents thoroughly by pipetting and spin down briefly.
SECTION: Step8. Store the MinION/GridION flow cell for later use
8.2. Rotate the flow cell priming port cover clockwise so that the priming port is visible.
SECTION: Step8. Store the MinION/GridION flow cell for later use
8.3. Check for air between the priming port and the sensor array. If necessary, using a P1000 draw back a small volume to remove any air (a few μls):
1. Set a P1000 pipette to 200 μl
2. Insert the tip into the priming port
3. Turn the wheel until the dial shows 220-230 μl, or until you can see a small volume of buffer/liquid entering the        pipette tip. 
4. Visually check that there is continuous buffer from the priming port across the sensor array.
SECTION: Step8. Store the MinION/GridION flow cell for later use
8.4. Slowly add 500 μl of Storage Buffer (S) through the priming port of the flow cell.
SECTION: Step8. Store the MinION/GridION flow cell for later use
8.5. Close the priming port.
SECTION: Step8. Store the MinION/GridION flow cell for later use
8.6. Using a P1000, remove all fluid from the waste channel through Waste port 1. As both the priming port and SpotON sample port are closed, no fluid should leave the sensor array area.
 

SECTION: Step8. Store the MinION/GridION flow cell for later use
8.7. The flow cell can now be stored at 4-8°C.
SECTION: Step8. Store the MinION/GridION flow cell for later use
8.8. When you wish to reuse the flow cell, remove the flow cell from storage, and allow it to warm to room temperature for ~5 minutes. You will need to perform a Flow Cell Check before loading the next library.