Oct 12, 2023

Public workspaceNanopore Sequencing with Flongle Flow Cells

DOI
dx.doi.org/10.17504/protocols.io.j8nlko3mwv5r/v1
  • 1National University of Singapore
NUS iGEM
Open access
DOI: dx.doi.org/10.17504/protocols.io.j8nlko3mwv5r/v1
Protocol Citation: NUS iGEM 2023. Nanopore Sequencing with Flongle Flow Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlko3mwv5r/v1
Manuscript citation:
Oxford Nanopore Technologies. (n.d.). Ligation sequencing amplicons - Native Barcoding Kit 24 V14 (SQK-NBD114.24). Retrieved October 12, 2023, from https://community.nanoporetech.com/docs/prepare/library_prep_protocols/ligation-sequencing-amplicons-native-barcoding-v14-sqk-nbd114-24/v/nba_9168_v114_revk_15sep2022
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: October 08, 2023
Last Modified: October 12, 2023
Protocol Integer ID: 88973
Keywords: Nanopore, Flongle, Flongle Flow Cells, Sequencing, Oligo Variants, Variation, Mutagenesis
Abstract
The 2023 NUS-Singapore iGEM Team followed this protocol to sequence their STAR oligo variants. The original protocol was released by Oxford Nanopore Technologies, but due to the use of a new type of flow cell in the experiment, some minor modifications to the steps were made to tailor it to our project.
Protocol materials
ReagentNEBNext Ultra II End Prep Reaction BufferNew England BiolabsCatalog #E7647
Step 2
ReagentAMPure XP Beads
In 7 steps
ReagentNEB Blunt/TA Ligase Master Mix Catalog #M0367
Step 21
ReagentQuick T4 DNA LigaseNew England BiolabsCatalog #E7180S
In 2 steps
ReagentNEBNext Ultra II End Repair/dA-Tailing Module - 24 rxnsNew England BiolabsCatalog #E7546S
Step 2
ReagentNEBNext Ultra II End Prep Enzyme MixNew England BiolabsCatalog #E7646
Step 2
Safety warnings
Attention
Proper lab PPE must be worn at all times.
End-prep
End-prep
18m
Mix ReagentAMPure XP BeadsContributed by users 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 ReagentNEBNext Ultra II End Repair/dA-Tailing Module - 24 rxnsNew England BiolabsCatalog #E7546S in accordance with the manufacturer's instructions, and place on ice. (ReagentNEBNext Ultra II End Prep Enzyme MixNew England BiolabsCatalog #E7646 do not vortex, ReagentNEBNext Ultra II End Prep Reaction BufferNew England BiolabsCatalog #E7647 vortex for at least Duration00:00:30 until no precipitate is visible.)

30s
Dilute the DNA Control Sample (DCS) by adding Amount105 µL Elution Buffer directly to one DCS tube. Mix gently by pipetting and spin down. (excess storage at Temperature-20 °C )

In clean 0.2 ml thin-walled PCR tubes (or a clean 96-well plate), aliquot 200 fmol (Amount130 ng for 1 kb amplicons) of DNA per sample.
Make up each sample to Amount11.5 µL 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 Amount2.5 µL to each well.
ItemVolume
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 Temperature20 °C for Duration00:05:00 and Temperature65 °C for Duration00:05:00 .

10m
Transfer each sample into a clean 1.5 ml Eppendorf DNA LoBind tube.
Resuspend ReagentAMPure XP BeadsContributed by users by vortexing.

Add Amount15 µL of resuspended ReagentAMPure XP BeadsContributed by users to each end-prep reaction and mix by flicking the tube.

Incubate on a Hula mixer (rotator mixer) for Duration00:05:00 at room temperature.

5m
Prepare Amount500 µL 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 Amount200 µL 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 Duration00:00:30 , but do not dry the pellets to the point of cracking.

30s
Remove the tubes from the magnetic rack and resuspend the pellet in 10 µl nuclease-free water. Spin down and incubate for Duration00:02:00 at room temperature.

2m
Pellet the beads on a magnet until the eluate is clear and colourless.
Remove and retain Amount10 µL of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube. Sample storage at Temperature4 °C .

Native Barcode Ligation
Native Barcode Ligation
47m 40s
Prepare ReagentNEB Blunt/TA Ligase Master Mix Contributed by usersCatalog #M0367 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:
ItemVolume
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 Duration00:20:00 at room temperature.
20m
Add the Amount2 µL 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; Amount22 µL per sample
Resuspend ReagentAMPure XP BeadsContributed by users by vortexing.
Add Amount9 µL ReagentAMPure XP BeadsContributed by users per sample to the pooled reaction, and mix by pipetting for a 0.4X clean.
Incubate on a Hula mixer (rotator mixer) for Duration00:10:00 at room temperature.
10m
Prepare Amount2 mL of fresh 80% ethanol in nuclease-free water.
Spin down the sample and pellet on a magnet for Duration00:05:00 . Keep the plate on the magnetic rack until the eluate is clear and colourless, and pipette off the supernatant.
5m
Keep the tube on the magnetic rack and wash the beads with Amount700 µL 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 for Duration00:00:30 , but do not dry the pellet to the point of cracking.
30s
Remove the tube from the magnetic rack and resuspend the pellet in Amount35 µL nuclease-free water by gently flicking.
Incubate for Duration00:10:00 at Temperature37 °C . Every Duration00:02:00 , agitate the sample by gently flicking for Duration00:00:10 to encourage DNA elution.
12m 10s
Pellet the beads on a magnetic rack until the eluate is clear and colourless. Remove and retain Amount35 µL of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
Adaptor Ligation and Clean-up
Adaptor Ligation and Clean-up
43m 10s
Prepare the NEBNext Quick Ligation Reaction Module according to the manufacturer's instructions, and place on ice. (Do NOT vortex ReagentQuick T4 DNA LigaseNew England BiolabsCatalog #E7180S .)
Spin down the Native Adapter (NA) and ReagentQuick T4 DNA LigaseNew England BiolabsCatalog #E7180S , 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:
ItemVolume
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 Duration00:20:00 at room temperature.

20m
Resuspend ReagentAMPure XP BeadsContributed by users by vortexing.

Add Amount20 µL of resuspended ReagentAMPure XP BeadsContributed by users to the reaction and mix by pipetting.
Incubate on a Hula mixer (rotator mixer) for Duration00:10:00 at room temperature.
10m
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 Amount125 µL 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 Amount7 µL Elution Buffer (EB).

Spin down and incubate for Duration00:10:00 at Temperature37 °C . Every Duration00:02:00 , agitate the sample by gently flicking for Duration00:00:10 to encourage DNA elution.
12m 10s
Pellet the beads on a magnet until the eluate is clear and colorless, for at least Duration00:01:00 .
1m
Remove and retain Amount7 µL of EB containing the DNA library into a clean 1.5 ml Eppendorf SNA LoBind tubes.
Quantify Amount1 µL of eluted sample using Nanodrop.
Make up the library to Amount5 µL at 5-10 fmol.
Loading the Flongle Flow Cell
Loading the Flongle Flow Cell
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 Amount117 µL of Flow Cell Flush(FCF) with Amount3 µL 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:
ItemVolume
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.
Protocol references
Oxford Nanopore Technologies. (n.d.). Ligation sequencing amplicons - Native Barcoding Kit 24 V14 (SQK-NBD114.24). Retrieved October 12, 2023, from https://community.nanoporetech.com/docs/prepare/library_prep_protocols/ligation-sequencing-amplicons-native-barcoding-v14-sqk-nbd114-24/v/nba_9168_v114_revk_15sep2022