May 25, 2023

Public workspaceNanopore genome sequencing with barcode

DOI
dx.doi.org/10.17504/protocols.io.dm6gpb2wjlzp/v1
  • 1KMU
Guan Jie Phang
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DOI: dx.doi.org/10.17504/protocols.io.dm6gpb2wjlzp/v1
Protocol Citation: Guan Jie Phang 2023. Nanopore genome sequencing with barcode. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpb2wjlzp/v1
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: May 29, 2022
Last Modified: September 13, 2023
Protocol Integer ID: 63437
Abstract
The modified version of official protocol using Kappa chemicals
Step1. End-prep & A-Tailing
Step1. End-prep & A-Tailing
1h
1h
In a 200μl PCR tube, mix the following:

ReagentVolumeCap color
40 fmol DNA 250ng/ sample25 μl
End Repair & A-Tailing Buffer (KAPA)3.5 μl Purple
End Repair & A-Tailing Enzyme Mix (KAPA)1.5 μl Purple
Total30 μl

Ensure the components are thoroughly mixed by pipetting, and spin down.
Using a thermal cycler, incubate at Temperature20 °C forDuration00:30:00 andTemperature65 °C for Duration00:30:00 . Hold atTemperature4 °C

1h
Step2. Barcode Ligation
Step2. Barcode Ligation
30m
30m
Thaw LNB, Native Barcode at room temperature, spin down and mix by vortex.

Note
Place on ice immediately after thawing and mixing.

In a 200μl PCR tube, mix in the following order:

ReagentVolumeColor
End repair and A-tailing reaction product30μl
Native Barcode2.5μlWhite
LNB (LSK109 Kit)13.75μlWhite
DNA Ligase (KAPA)5μlYellow
DI Water3.75μl
Total55μl

Ensure the components are thoroughly mixed by pipetting, and spin down.
Using a thermal cycler, incubate at Temperature20 °C Duration00:30:00 .

Note
Prewarm HyperPure beads at RT before use.

Note
To achieve higher conversion rates and library yields, particularly for low-input samples, consider increasing the ligation time—to a maximum of 4 hrs at 20°C, then overnight at 2°C to 8°C. Please note that longer ligation times may lead to increased levels of adapter-dimer. Adapter concentrations may have to be optimized if ligation times are extended significantly.

30m
Step3. Barcode Ligation Products clean-up
Step3. Barcode Ligation Products clean-up
30m
30m
Add 44 ul (0.8X) of resuspended HyperPure beads to the barcode-ligated reaction and mix by flicking the tube.
Incubate on a Hula mixer (rotator mixer) for 5 minutes at RT.
Prepare 500 ul of fresh 70% ethanol in Nuclease-free water.
Spin down the sample and pellet on a magnetic stand.
Keep the tube on the magnetic stand, and pipette off the supernatant.
Keep the tube on the magnetic stand and wash the beads with 200 ul of freshly-prepared 70% ethanol without disturbing the pellet.
Remove the ethanol using a pipette and discard.
Repeat the previous step to remove ethanol.
Spin down and place the tube back on the magnetic stand.
Pipette off any residual ethanol.
Allow to dry for ~30 seconds(1-3 min), but do not dry the pellet to the point of cracking.
Remove the tube from the magnetic stand and resuspend the pellet in 11 ul Nuclease-free water.(Temperature55 °C 1 min)

Pellet the beads on a magnetic stand until the eluate is clear and colourless.
Remove and retain 11 ul of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
Quantify 1 μl of eluted sample using a Quantus fluorometer.


Step4. Adapter Ligation (AMII)
Step4. Adapter Ligation (AMII)
30m
30m
Pool samples to be totaly 100 fmol DNA mix.

ReagentVolumeColor
~50 fmol pooled barcoded DNA in DDW67.5 μl
Adapter Mix II (AMII)5 μlGreen
LNB(LSK109 Kit)27.5 μlWhite
DNA Ligase (KAPA)10 μlYellow
Total volume110 μl

Using a thermal cycler, incubate at Temperature20 °C Duration00:30:00 .
Note
Prewarm HyperPure beads at RT before use.


Note
To achieve higher conversion rates and library yields, particularly for low-input samples, consider increasing the ligation time—to a maximum of 4 hrs at 20°C, or after 15 min incubation and then overnight at 2°C to 8°C. Please note that longer ligation times may lead to increased levels of adapter-dimer. Adapter concentrations may have to be optimized if ligation times are extended significantly.

30m
Step5. DNA library clean-up
Step5. DNA library clean-up
30m
30m
Add 44 ul (0.4x beads) of resuspended HyperPuer beads to the adapter-ligated reaction and mix by pipetting.
Incubate on a Hula mixer (rotator mixer) for 5 minutes at RT.
Place on magnetic stand, allow beads to pellet and pipette off supernatant.
Wash the beads by adding either 250 μl Long Fragment Buffer (LFB) or 250 μl S Fragment Buffer (SFB)(<3k).
Flick the beads to resuspend, then return the tube to the magnetic stand and allow the beads to pellet.
Remove the supernatant using a pipette and discard.
Wash the beads by adding either 250 μl Long Fragment Buffer (LFB) or 250 μl S Fragment Buffer (SFB)(<3k).
Flick the beads to resuspend, then return the tube to the magnetic stand and allow the beads to pellet.
Remove the supernatant using a pipette and discard.
Spin down and place the tube back on the magnetic stand.
Pipette off any residual supernatant.
Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
Remove the tube from the magnetic stand and resuspend the pellet in 14 ul Elution Buffer.
Keep at RT for 10 min.
Pellet the beads on a magnetic stand until the eluate is clear and colourless.
Remove and retain 14 ul of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
Quantify 1 μl of eluted sample using a Quantus fluorometer.
Note
Loading 5–50 fmol (or less than 300ng) of final prepared library onto R9.4.1 flow cells.

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

Note
Press down firmly on the flow cell to ensure correct thermal and electrical contact.

Check flow cell (Check the pore)

Note
This takes about 10 minutes.

Mix the Sequencing Buffer (SQB), Flush Tether (FLT) and Flush Buffer (FB) tubes by pipetting and spin down at room temperature.
Remove the flow cell from the machine and slide the priming port cover clockwise to open the priming port.
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.

Note
Visually check that there is continuous buffer from the priming port across the sensor array.

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.

ReagentVolumeColor
Flush Tether (FLT)30μlPurple
Flush Buffer (FB)New oneBlue
Total1.2ml

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.
Note
Closed the priming port.

Thoroughly mix the contents of the Loading Beads (LB) by flick.
In a new tube, prepare the library for loading as follows:
ReagentVolumeColor
Sequencing Buffer (SQB)37.5μlRed
Loading Beads (LB),mixed immediately before use25.5μlPink
DNA library12μl
Total75μl

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.
Note
Load the library as soon as possible after this step.

Mix the prepared library gently by pipetting up and down just prior to loading.
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.
Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the priming port and replace the GridION.
Step7.Flow Cell Wash
Step7.Flow Cell Wash
30m
30m
Place the tube of Wash Mix (WMX) on ice. Do not vortex the tube.
Thaw one tube of Wash Diluent (DIL) at room temperature.
Mix the contents of Wash Diluent (DIL) thoroughly by vortexing, spin down briefly and place on ice.
In a clean 1.5 ml Eppendorf DNA tube, prepare the following Flow Cell Wash Mix:

ComponentVolumeColor
Wash Mix (WMX)2μlBrown
Wash Diluent (DIL)398μlBrown
Total400μl

Mix well by pipetting, and place on ice. Do not vortex the tube.
Stop or pause the sequencing experiment in MinKNOW, and leave the flow cell in the device.
Ensure that the priming port cover and SpotON sample port cover are in the positions indicated in the figure below.
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.
Rotate the flow cell priming port cover clockwise so that the priming port is visible.
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.
Load 400 μl of the prepared Flow Cell Wash Mix into the flow cell via the priming port, avoiding the introduction of air.
Close the priming port and wait for 30 minutes.
Ensure that the priming port cover and SpotON sample port cover are in the positions indicated in the figure below.
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.

Note
It is vital that the flow cell priming port and SpotON sample port are closed to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.

Step8. Store the MinION/GridION flow cell for later use
Step8. Store the MinION/GridION flow cell for later use
30m
30m
Thaw one tube of Storage Buffer (S) at room temperature.
Mix contents thoroughly by pipetting and spin down briefly.
Rotate the flow cell priming port cover clockwise so that the priming port is visible.
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.
Slowly add 500 μl of Storage Buffer (S) through the priming port of the flow cell.
Close the priming port.
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.
Note
It is vital that the flow cell priming port and SpotON sample port are closed to prevent air from being drawn across the sensor array area, which would lead to a significant loss of sequencing channels.

The flow cell can now be stored at 4-8°C.
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.
Note
We recommend storing libraries in Eppendorf DNA LoBind tubes at 4°C for short term storage or repeated use, for example, re-loading flow cells between washes. For single use and long term storage of more than 3 months, we recommend storing libraries at -80°C in Eppendorf DNA LoBind tubes. For further information, please refer to the DNA library stability Know-How document.