Nanopore Sequencing with Flongle Flow Cells

Mix and DNA Control Sample (DCS) at room temperature by vortexing. Keep the beads at room temperature and store the DNA Control Sample (DCS) on ice.

Prepare the in accordance with the manufacturer's instructions, and place on ice. ( do not vortex, vortex for at least0h 0m 30s until no precipitate is visible.)

Dilute the DNA Control Sample (DCS) by adding 105µL Elution Buffer directly to one DCS tube. Mix gently by pipetting and spin down. (excess storage at -20°C)

In clean 0.2 ml thin-walled PCR tubes (or a clean 96-well plate), aliquot 200 fmol (130ng for 1 kb amplicons) of DNA per sample.

Make up each sample to 11.5µL using nuclease-free water. Mix gently by pipetting and spin down.

Combine the following components per tube/well:

Between each addition, pipette mix 10 - 20 times.

Recommend to prepare a master mix and add 2.5µL to each well.

A	B
200 fmol (130 ng for 1 kb amplicons) amplicon DNA	11.5μL
Diluted DNA Control Sample (DCS)	1μL
Ultra II End-prep Reaction Buffer	1.75μL
Ultra II End-prep Enzyme Mix	0.75μL

Ensure the components are thoroughly mixed by pipetting and spin down in a centrifuge.

Using a thermal cycler, incubate at 20°C for 0h 5m 0s and 65°C for 0h 5m 0s.

Transfer each sample into a clean 1.5 ml Eppendorf DNA LoBind tube.

Resuspend by vortexing.

Add 15µL of resuspended to each end-prep reaction and mix by flicking the tube.

Incubate on a Hula mixer (rotator mixer) for 0h 5m 0s at room temperature.

Prepare 500µL of fresh 80% ethanol in nuclease-free water.

Spin down the samples and pellet the beads on a magnet until the eluate is clear and colourless. Keep the tubes on the magnet and pipette off the supernatant.

 Keep the tube on the magnet and wash the beads with 200µL of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

Repeat step 15.

Briefly spin down and place the tubes back on the magnet for the beads to pellet. Pipette off any residual ethanol. Allow to dry for 0h 0m 30s, but do not dry the pellets to the point

of cracking.

Remove the tubes from the magnetic rack and resuspend the pellet in 10 µl nuclease-free water. Spin down and incubate for 0h 2m 0s at room temperature.

Pellet the beads on a magnet until the eluate is clear and colourless.

Remove and retain 10µL of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube. Sample storage at 4°C.

Prepare according to the manufacturer's instructions, and place on ice.

Thaw the EDTA at room temperature and mix by vortexing. Then spin down and place on ice.

Thaw the Native Barcodes (NB01-24) required for the number of samples at room temperature. Individually mix the barcodes by pipetting, spin down, and place them on ice.

Select a unique barcode for each sample to be run together on the same flow cell. Up to 24 samples can be barcoded and combined in one experiment. (only use one barcode per sample)

In clean 0.2 ml PCR-tubes or a 96-well plate, add the reagents in the following order per well:

A	B
End-prepped DNA	7.5μL
Native Barcode (NB01-24)	2.5μL
Blunt/TA Ligase Master Mix	10μL

Ensure the reaction is thoroughly mixed by gently pipetting and spin down briefly.

Incubate for 0h 20m 0s at room temperature.

Add the 2µL of clear cap EDTA to each well and mix thoroughly by pipetting and spin down briefly.

Pool all the barcoded samples in a 1.5 ml Eppendorf DNA LoBind tube; 22µL per sample

Resuspend by vortexing.

Add 9µL per sample to the pooled reaction, and mix by pipetting for a 0.4X clean.

Incubate on a Hula mixer (rotator mixer) for 0h 10m 0s at room temperature.

Prepare 2mL of fresh 80% ethanol in nuclease-free water.

Spin down the sample and pellet on a magnet for 0h 5m 0s. Keep the plate on the magnetic rack until the eluate is clear and colourless, and pipette off the supernatant.

Keep the tube on the magnetic rack and wash the beads with 700µL of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

Repeat step 35.

Spin down and place the tube back on the magnetic rack. Pipette off any residual ethanol.

Allow the pellet to dry for0h 0m 30s , but do not dry the pellet to the point of cracking.

Remove the tube from the magnetic rack and resuspend the pellet in 35µL nuclease-free water by gently flicking.

Incubate for 0h 10m 0s at 37°C. Every 0h 2m 0s, agitate the sample by gently flicking for 0h 0m 10s to encourage DNA elution.

Pellet the beads on a magnetic rack until the eluate is clear and colourless. Remove and retain 35µLof eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

Prepare the NEBNext Quick Ligation Reaction Module according to the manufacturer's instructions, and place on ice. (Do NOT vortex .)

Spin down the Native Adapter (NA) and , pipette mix and place on ice.

Thaw Elution Buffer at room temperature and mix by vortexing. Then spin down and place on ice.

Thaw either Long Fragment Buffer (LFB) >3kb or Short Fragment Buffer (SFB) <3kb at room temperature and mix by vortexing. Then spin down and place on ice.

In a 1.5 ml Eppendorf LoBind tube, mix in the following order:

A	B
Pooled barcoded sample	30μL
Native Adapter (NA)	5μL
NEBNext Quick Ligation Reaction Buffer(5X)	10μL
Quick T4 DNA Ligase	5μL

Ensure the reaction is thoroughly mixed by gently pipetting and spin down briefly.

Incubate the reaction for 0h 20m 0s at room temperature.

Resuspend by vortexing.

Add 20µL of resuspended to the reaction and mix by pipetting.

Incubate on a Hula mixer (rotator mixer) for 0h 10m 0s at room temperature.

Spin down the sample and pellet on the magnetic rack. Keep the tube on the magnet and pipette off the supernatant.

Wash the beads by adding either 125µL Long Fragment Buffer (LFB) or Short Fragment Buffer (SFB). Flick the beads to resuspend, spin down, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard.

Repeat Step 52.

Spin down and place the tube back on the magnet. Pipette off any residual supernatant.

Remove the tube from the magnetic rack and resuspend pellet in 7µL Elution Buffer (EB).

Spin down and incubate for 0h 10m 0s at 37°C. Every 0h 2m 0s, agitate the sample by gently flicking for 0h 0m 10s to encourage DNA elution.

Pellet the beads on a magnet until the eluate is clear and colorless, for at least 0h 1m 0s.

Remove and retain 7µL of EB containing the DNA library into a clean 1.5 ml Eppendorf SNA LoBind tubes.

Quantify 1µL of eluted sample using Nanodrop.

Make up the library to 5µL at 5-10 fmol.

Thaw the Sequencing Buffer(SB), Library Beads(LIB), Flow Cell Tether(FCT) and one tube of Flow Cell Flush(FCF) at room temperature before mixing by vortexing. Then spin down and

store on ice.

In a fresh 1.5 ml Eppendorf DNA LoBind tube, mix 117µL of Flow Cell Flush(FCF) with 3µL of Flow Cell Tether(FCT) and mix by pipetting.

Place the Flongle adaptor into the MinION or one of the five GridION positions.

Place the flow cell into Flonge adapter, and press the flow cell down until hearing a click.

Peel back the seal tab from the Flongle flow cell, up to a point where the sample port is exposed. Lift up the seal tab, pull the seal tab to open access to the sample port, then hold the seal tab open by using adhesive on the tab to stick to the MinION Mk1B lid.

To prime the flow cell with the mix of Flow Cell Flush(FSF) and Flow Cell Tether(FCT) that was prepared earlier, ensure that there is no air gap in the sample port or the pipette tip.

Place the P200 pipette tip inside the sample port and slowly dispense the priming fluid into the Flonge flow cell by slowly pipetting down. Recommend twisting the pipette plunger down to avoid flushing the flow cell too vigorously.

Vortex the vial of Library Beads(LIB). Immediately prepare the sequencing Mix in a fresh 1.5 ml        Eppendorf DNA LoBind tube for loading the Flongle as follows:

A	B
Sequencing Buffer(SB)	15μL
Library Beads(LIB) mixed immediately before use	10μL
DNA Library	5μL

To add the sequencing mix to the flow cell, ensure that there is no air gap in the sample port or the

pipette tip.

Place the P200 tip inside the sample port and slowly dispense the sequencing mix into the flow cell by slowly pipetting down. Recommend twisting the pipette plunger down to avoid flushing the flow cell too vigorously.

Seal the Flonge flow cell using the adhesive on the seal tab: stick the transparent adhesive tape to the sample port, replace the top(wheel icon section) of the seal tab to its original position.

Replace the sequencing platform lid and start sequencing.