Nanopore genome sequencing with barcode

In a 200μl PCR tube, mix the following:

A	B	C
40 fmol DNA 250ng/ sample	25 μl
End Repair & A-Tailing Buffer (KAPA)	3.5 μl	Purple
End Repair & A-Tailing Enzyme Mix (KAPA)	1.5 μl	Purple
Total	30 μl

Ensure the components are thoroughly mixed by pipetting, and spin down.

Using a thermal cycler, incubate at 20°C for0h 30m 0s and65°C for 0h 30m 0s. Hold at4°C

Thaw LNB, Native Barcode at room temperature, spin down and mix by vortex.

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

A	B	C
End repair and A-tailing reaction product	30μl
Native Barcode	2.5μl	White
LNB (LSK109 Kit)	13.75μl	White
DNA Ligase (KAPA)	5μl	Yellow
DI Water	3.75μl
Total	55μl

Ensure the components are thoroughly mixed by pipetting, and spin down.

Using a thermal cycler, incubate at 20°C``0h 30m 0s .

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.

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.(55°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.

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.

Pool samples to be totaly 100 fmol DNA mix.

A	B	C
~50 fmol pooled barcoded DNA in DDW	67.5 μl
Adapter Mix II (AMII)	5 μl	Green
LNB(LSK109 Kit)	27.5 μl	White
DNA Ligase (KAPA)	10 μl	Yellow
Total volume	110 μl

Using a thermal cycler, incubate at 20°C``0h 30m 0s .

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.

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.

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.

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.

In a new tube, prepare the library for loading as follows:

A	B	C
Sequencing Buffer (SQB)	37.5μl	Red
Loading Beads (LB),mixed immediately before use	25.5μl	Pink
DNA library	12μl
Total	75μl

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.

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.

Open the GridION and slide the flow cell under the clip.

Check flow cell (Check the pore)

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.

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.

A	B	C
Flush Tether (FLT)	30μl	Purple
Flush Buffer (FB)	New one	Blue
Total	1.2ml

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.

Thoroughly mix the contents of the Loading Beads (LB) by flick.

Place the tube of Wash Mix (WMX) on ice. Do not vortex the tube.

Thaw one tube of Wash Diluent (DIL) at room temperature.

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.

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:

A	B	C
Wash Mix (WMX)	2μl	Brown
Wash Diluent (DIL)	398μl	Brown
Total	400μl

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.

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.

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.