Ligation sequencing gDNA - Native Barcoding Kit 24 V14 (SQK-NBD114.24)

Version: NBE_9169_v114_revQ_15Sep2022 | Last update: 16/02/2024

Flow Cell Number: ......................................................................................

DNA Samples: .........................................................................................

Before start checklist

Materials	Consumables	Equipment
Native Barcoding Kit 24 V14 (SQK-NBD114.24)	NEB Blunt/TA Ligase Master Mix (NEB, M0367)	Hula mixer (gentle rotator mixer)
400 ng gDNA per sample if using >4 barcodes	NEBNext FFPE Repair Mix (NEB, M6630)	Microplate centrifuge, e.g. Fisherbrand™ Mini Plate Spinner Centrifuge (Fisher Scientific, 11766427)
OR 1000 ng gDNA per sample if using ≤4 barcodes	NEBNext Ultra II End repair/dA-tailing Module (NEB, E7546)	Microfuge
	NEBNext Quick Ligation Module (NEB, E6056)	Magnetic rack
	Eppendorf twin.tec® PCR plate 96 LoBind, semi-skirted (Eppendorf™, cat # 0030129504) with heat seals	Vortex mixer
	1.5 ml Eppendorf DNA LoBind tubes	Thermal cycler
	2 ml Eppendorf DNA LoBind tubes	Ice bucket with ice
	Nuclease-free water (e.g. ThermoFisher, AM9937)	Timer
	Freshly prepared 80% ethanol in nuclease-free water	Eppendorf 5424 centrifuge (or equivalent)
	Qubit™ Assay Tubes (Invitrogen, Q32856)	Qubit fluorometer (or equivalent for QC check)
	Qubit dsDNA HS Assay Kit (ThermoFisher, cat # Q32851)	Pipettes and pipette tips P2, P10, P20, P100, P200, P1000, Multichannel

INSTRUCTIONS

DNA repair and end-prep

Thaw the AMPure XP Beads (AXP) and DNA Control Sample (DCS) at RT and mix by vortexing. Keep the beads at RT and store the DNA Control Sample (DCS) on ice.
Prepare the NEBNext FFPE DNA Repair Mix and NEBNext Ultra II End Repair / dA-tailing Module reagents in accordance with manufacturer's instructions, and place on ice.
Thaw all reagents on ice. Flick and/or invert the reagent tubes to ensure they are well mixed. Note: Do not vortex the FFPE DNA Repair Mix or Ultra II End Prep Enzyme Mix.
Always spin down tubes before opening for the first time each day.
The Ultra II End Prep Buffer and FFPE DNA Repair Buffer may have a little precipitate. Allow the mixture to come to RT and pipette the buffer up and down several times to break up the precipitate, followed by vortexing the tube for 30 seconds to solubilise any precipitate. Note: It is important the buffers are mixed well by vortexing. The FFPE DNA Repair Buffer may have a yellow tinge and is fine to use if yellow.
IMPORTANT: Do not vortex the NEBNext FFPE DNA Repair Mix or NEBNext Ultra II End Prep Enzyme Mix.
IMPORTANT: It is important that the NEBNext FFPE DNA Repair Buffer and NEBNext Ultra II End Prep Reaction Buffer are mixed well by vortexing.
Dilute your DNA Control Sample (DCS) by adding 105 µl Elution Buffer (EB) directly to one DCS tube. Mix gently by pipetting and spin down.
In clean 0.2 ml thin-walled PCR tubes (or a clean 96-well plate), prepare your DNA samples: For >4 barcodes, aliquot 400 ng per sample. For ≤4 barcodes, aliquot 1000 ng per sample.
Make up each sample to 11 µl using Nuclease-free water. Mix gently by pipetting and spin down.
Combine the following components per tube/well: 11 µl DNA sample, 1 µl Diluted DNA Control Sample (DCS), 0.875 µl NEBNext FFPE DNA Repair Buffer, 0.875 µl Ultra II End-prep Reaction Buffer, 0.75 µl Ultra II End-prep Enzyme Mix, 0.5 µl NEBNext FFPE DNA Repair Mix.
Ensure the components are thoroughly mixed by pipetting and spin down in a centrifuge.
Using a thermal cycler, incubate at 20°C for 5 minutes and 65°C for 5 minutes.
Transfer each sample into a clean 1.5 ml Eppendorf DNA LoBind tube.
Resuspend the AMPure XP beads (AXP) by vortexing.
Add 15 µl of resuspended AMPure XP Beads (AXP) to each end-prep reaction and mix by flicking the tube.
Incubate on a Hula mixer (rotator mixer) for 5 minutes at RT.
Prepare sufficient fresh 80% ethanol in Nuclease-free water for all of your samples. Allow enough for 400 µl per sample, with some excess.
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 200 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
Repeat the previous step.
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 30 seconds, 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 2 minutes at RT.
Pellet the beads on a magnet until the eluate is clear and colourless.
Remove and retain 10 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube. Dispose of the pelleted beads.
Quantify 1 µl of each eluted sample using a Qubit fluorometer.
Take forward an equimolar mass of each sample to be barcoded forward into the native barcode ligation step. However, you may store the samples at 4°C overnight.

Native barcode ligation

Prepare the NEB Blunt/TA Ligase Master Mix according to the manufacturer's instructions, and place on ice: Thaw the reagents at RT. Spin down the reagent tubes for 5 seconds. Ensure the reagents are fully mixed by performing 10 full volume pipette mixes.
Thaw the EDTA at RT and mix by vortexing. Then spin down and place on ice.
Thaw the Native Barcodes (NB01-24) required for your number of samples at RT. 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.
In clean 0.2 ml PCR-tubes or a 96-well plate, add the reagents in the following order per well: 7.5 µl End-prepped DNA, 2.5 µl Native Barcode (NB01-24), 10 µl Blunt/TA Ligase Master Mix.
Thoroughly mix the reaction by gently pipetting and briefly spinning down.
Incubate for 20 minutes at RT.
Add the following volume of EDTA to each well and mix thoroughly by pipetting and spin down briefly: 2 µl For clear cap EDTA, 4 µl For blue cap EDTA.
Pool all the barcoded samples in a 1.5 ml Eppendorf DNA LoBind tube.
Resuspend the AMPure XP Beads (AXP) by vortexing.
Add 0.4X AMPure XP Beads (AXP) to the pooled reaction, and mix by pipetting.
Incubate on a Hula mixer (rotator mixer) for 10 minutes at RT.
Prepare 2 ml of fresh 80% ethanol in Nuclease-free water.
Spin down the sample and pellet on a magnet for 5 minutes. Keep the tube 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 700 µl of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
Repeat the previous step.
Spin down and place the tube back on the magnetic rack. Pipette off any residual ethanol. Allow the pellet to dry for ~30 seconds, but do not dry the pellet to the point of cracking.
Remove the tube from the magnetic rack and resuspend the pellet in 35 µl Nuclease-free water by gently flicking.
Incubate for 10 minutes at 37°C. Every 2 minutes, agitate the sample by gently flicking for 10 seconds to encourage DNA elution.
Pellet the beads on a magnetic rack until the eluate is clear and colourless.
Remove and retain 35 µl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.
Quantify 1 µl of eluted sample using a Qubit fluorometer.
Take forward the barcoded DNA library to the adapter ligation and clean-up step. However, you may store the sample at 4°C overnight.

Adapter ligation and clean-up

Prepare the NEBNext Quick Ligation Reaction Module according to the manufacturer's instructions, and place on ice: Thaw the reagents at RT. Spin down the reagent tubes for 5 seconds. Ensure the reagents are fully mixed by performing 10 full volume pipette mixes. Note: Do NOT vortex the Quick T4 DNA Ligase.
IMPORTANT: Do not vortex the Quick T4 DNA Ligase.
Spin down the Native Adapter (NA) and Quick T4 DNA Ligase, pipette mix and place on ice.
Thaw the Elution Buffer (EB) at RT and mix by vortexing. Then spin down and place on ice.
IMPORTANT: Depending on the wash buffer (LFB or SFB) used, the clean-up step after adapter ligation is designed to either enrich for DNA fragments of >3 kb, or purify all fragments equally. To enrich for DNA fragments of 3 kb or longer, use Long Fragment Buffer (LFB). To retain DNA fragments of all sizes, use Short Fragment Buffer (SFB).
Thaw either Long Fragment Buffer (LFB) or Short Fragment Buffer (SFB) at RT and mix by vortexing. Then spin down and place on ice.
In a 1.5 ml Eppendorf LoBind tube, mix in the following order: 30 µl Pooled barcoded sample, 5 µl Native Adapter (NA), 10 µl NEBNext Quick Ligation Reaction Buffer (5X), 5 µl Quick T4 DNA Ligase.
Thoroughly mix the reaction by gently pipetting and briefly spinning down.
Incubate the reaction for 20 minutes at RT.
IMPORTANT: The next clean-up step uses Long Fragment Buffer (LFB) or Short Fragment Buffer (SFB) rather than 80% ethanol to wash the beads. The use of ethanol will be detrimental to the sequencing reaction.
Resuspend the AMPure XP Beads (AXP) by vortexing.
Add 20 µl of resuspended AMPure XP Beads (AXP) to the reaction and mix by pipetting.
Incubate on a Hula mixer (rotator mixer) for 10 minutes at RT.
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 125 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 the previous step.
Spin down and place the tube back on the magnet. 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 rack and resuspend the pellet in 25 µl Elution Buffer (EB).
Spin down and incubate for 10 minutes at 37°C. Every 2 minutes, agitate the sample by gently flicking for 10 seconds to encourage DNA elution.
Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
Remove and retain 25 µl of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube.
Quantify 1 µl of eluted sample using a Qubit fluorometer.
Depending on your required data output, prepare your final library to 35-50 fmol for high output of simplex data, or 10-20 fmol for duplex data, in 32 µl of Elution Buffer (EB).
The prepared library is used for loading into the flow cell. Store the library on ice or at 4°C until ready to load.

Priming and loading the PromethION Flow Cell

IMPORTANT: This kit is only compatible with R10.4.1 flow cells (FLO-PRO114M).
Thaw the Sequencing Buffer (SB), Library Beads (LIB) or Library Solution (LIS, if using), Flow Cell Tether (FCT) and Flow Cell Flush (FCF) at RT before mixing by vortexing. Then spin down and store on ice.
To prepare the flow cell priming mix, combine Flow Cell Tether (FCT) and Flow Cell Flush (FCF), as directed below. Mix by vortexing at RT.
- 1,170 µl Flow Cell Flush (FCF)
- 30 µl Flow Cell Tether (FCT)
IMPORTANT: After taking flow cells out of the fridge, wait 20 minutes before inserting the flow cell into the PromethION for the flow cell to come to RT. Condensation can form on the flow cell in humid environments. Inspect the gold connector pins on the top and underside of the flow cell for condensation and wipe off with a lint-free wipe if any is observed. Ensure the heat pad (black pad) is present on the underside of the flow cell.
For PromethION 2 Solo, load the flow cell(s) as follows: Place the flow cell flat on the metal plate. Slide the flow cell into the docking port until the gold pins or green board cannot be seen.
For the PromethION 24/48, load the flow cell(s) into the docking ports: Line up the flow cell with the connector horizontally and vertically before smoothly inserting into position. Press down firmly onto the flow cell and ensure the latch engages and clicks into place.
IMPORTANT: Insertion of the flow cells at the wrong angle can cause damage to the pins on the PromethION and affect your sequencing results. If you find the pins on a PromethION position are damaged, please contact support@nanoporetech.com for assistance.
Slide the inlet port cover clockwise to open.
IMPORTANT: Take care when drawing back buffer from the flow cell. Do not remove more than 20-30 µl, and make sure that the array of pores are covered by buffer at all times. Introducing air bubbles into the array can irreversibly damage pores.
After opening the inlet port, draw back a small volume to remove any air bubbles: Set a P1000 pipette tip to 200 µl. Insert the tip into the inlet port. Turn the wheel until the dial shows 220-230 µl, or until you see a small volume of buffer entering the pipette tip.
Load 500 µl of the priming mix into the flow cell via the inlet port, avoiding the introduction of air bubbles. Wait five minutes. During this time, prepare the library for loading using the next steps in the protocol.
Thoroughly mix the contents of the Library Beads (LIB) by pipetting.
IMPORTANT: The Library Beads (LIB) tube contains a suspension of beads. These beads settle very quickly. It is vital that they are mixed immediately before use.
In a new 1.5 ml Eppendorf DNA LoBind tube, prepare the library for loading as follows: 100 µl Sequencing Buffer (SB), 68 µl Library Beads (LIB) thoroughly mixed before use, or Library Solution (LIS), 32 µl DNA library.
Complete the flow cell priming by slowly loading 500 µl of the priming mix into the inlet port.
Mix the prepared library gently by pipetting up and down just prior to loading.
Load 200 µl of library into the inlet port using a P1000 pipette.
Close the valve to seal the inlet port.
IMPORTANT: Install the light shield on your flow cell as soon as library has been loaded for optimal sequencing output.
If the light shield has been removed from the flow cell, install the light shield as follows: Align the inlet port cut out of the light shield with the inlet port cover on the flow cell. The leading edge of the light shield should sit above the flow cell ID. Firmly press the light shield around the inlet port cover. The inlet port clip will click into place underneath the inlet port cover.
Close the PromethION lid when ready to start a sequencing run on MinKNOW.

Flow cell reuse and returns

After your sequencing experiment is complete, if you would like to reuse the flow cell, please follow the Flow Cell Wash Kit protocol and store the washed flow cell at 2-8°C.
Alternatively, follow the returns procedure to flush out the flow cell ready to send back to Oxford Nanopore.
IMPORTANT: If you encounter issues or have questions about your sequencing experiment, please refer to the Troubleshooting Guide that can be found in the online version of this protocol.

	Ligation Sequencing gDNA - Native Barcoding Kit 24 V14 Protocol Detailed protocol for preparing genomic DNA (gDNA) for sequencing using the Oxford Nanopore Technologies Native Barcoding Kit 24 V14 (SQK-NBD114.24). Covers DNA repair, end-prep, ligation, clean-up, and flow cell preparation steps.
	Ligation Sequencing DNA V14 (SQK-LSK114) Protocol A comprehensive protocol for Ligation Sequencing DNA V14 (SQK-LSK114) by Oxford Nanopore Technologies, detailing steps for DNA repair, end-prep, adapter ligation, library preparation, and flow cell setup for MinION and GridION devices.
	Oxford Nanopore Large Cohort Sequencing Workflow for Clinical Research Accelerate clinical research with Oxford Nanopore's PromethION 24 platform. This guide details a comprehensive workflow for large cohort genomic and epigenomic analysis, from DNA extraction to variant calling.
	Direct RNA Sequencing Workflow for Isoform and Modification Analysis \| Oxford Nanopore Technologies Explore the PCR-free, direct RNA sequencing workflow from Oxford Nanopore Technologies for accurate detection of RNA isoforms, gene expression, and RNA modifications like m6A methylation. Learn about extraction, library preparation, sequencing, and analysis.
	Rapid Whole-Genome Sequencing Workflow for Variant and Methylation Identification \| Oxford Nanopore Technologies An overview of the Oxford Nanopore Technologies Rapid WGS workflow for fast, end-to-end identification of pathogenic variants and methylation from blood samples using the PromethION 24. This PCR-free nanopore sequencing approach enables rapid turnaround times, comprehensive variant calling (SNVs, SVs, repeat expansions), and epigenetic analysis from a single dataset.
	Ligation Sequencing gDNA V14 - Lambda Control Protocol Detailed protocol for Oxford Nanopore Technologies' Ligation Sequencing Kit (SQK-LSK114) using gDNA and a Lambda control, covering DNA repair, end-prep, adapter ligation, and flow cell preparation.
	PromethION 2 Solo Quick Start Guide A quick start guide for setting up and verifying the PromethION 2 Solo device, including pre-installation checks, device setup, hardware checks, and power-off procedures.
	Oxford Nanopore Service Provider Programme: Transform Services with Multiomic Insights Learn how Oxford Nanopore's Service Provider Programme empowers labs to deliver advanced multiomic insights, attract service projects, and scale innovation with their cutting-edge sequencing technology and EPI2ME platform.