Oxford Nanopore Technologies (ONT) library preparation and sequencing of DNA prepared using droplet Multiple Displacement Amplification (dMDA)

Nadine Holmes; Ester Kalef-Ezra; Christos Proukakis

Jul 30, 2024

Oxford Nanopore Technologies (ONT) library preparation and sequencing of DNA prepared using droplet Multiple Displacement Amplification (dMDA)

DOI

dx.doi.org/10.17504/protocols.io.q26g71x2qgwz/v1

Nadine Holmes¹,
Ester Kalef-Ezra^2,3,
Christos Proukakis^2,3

¹DeepSeq, School of Life Sciences, University of Nottingham, Nottingham, UK;
²Department of Clinical and Movement Neurosciences, Queen Square Institute of Neurology, University College London, London, UK;
³Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815

Nadine Holmes: Nadine Holmes: nadine.holmes1@nottingham.ac.uk;
Ester Kalef-Ezra: Ester Kalef-Ezra: e.kalef-ezra@ucl.ac.uk;
Christos Proukakis: Christos Proukakis: c.proukakis@ucl.ac.uk

University College London

Ester Kalef-Ezra

University College London

DOI: dx.doi.org/10.17504/protocols.io.q26g71x2qgwz/v1

Protocol Citation: Nadine Holmes, Ester Kalef-Ezra, Christos Proukakis 2024. Oxford Nanopore Technologies (ONT) library preparation and sequencing of DNA prepared using droplet Multiple Displacement Amplification (dMDA). protocols.io https://dx.doi.org/10.17504/protocols.io.q26g71x2qgwz/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: July 04, 2024

Last Modified: July 30, 2024

Protocol Integer ID: 103055

Keywords: ASAPCRN

Funders Acknowledgements:

This research was funded in whole by Aligning Science Across Parkinson’s [Grant ID: 000430] through the Michael J. Fox Foundation for Parkinson’s Research (MJFF).

Grant ID: Grant ID: 000430

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Abstract

Debranching of DNA generated by Multiple Displacement Amplification (MDA) is an essential step prior to nanopore sequencing. This protocol describes an optimised T7-based debranching protocol for genomic DNA amplified from single nuclei using droplet MDA (dMDA) obtained according to Kalef-Ezra et al 2023 (https://www.protocols.io/view/manual-isolation-of-nuclei-from-human-brain-using-kxygxzjjov8j/v1). Subsequent library preparation and sequencing steps are also outlined here.

Materials

ABCDE
ItemSupplierCatalogue NumberPreparation prior to useStorage
Nuclease-free waterSigma AldrichW4502AliquotRT
T7 Endonuclease I (10U/ul)NEBM0302SPlace on ice-20 °C
10X NEBuffer 2NEBM0302SThaw on ice-20 °C
AMPure XP BeadsBeckman CoulterA63882Equilibrate at RT for 30 min4 °C
1 M Tris-HCl pH 8Thermo ScientificJ22638-AERT
0.5 M EDTA pH 8, nuclease-freeThermo Fisher ScientificAM9260GRT
5M NaClThermo Fisher ScientificAM9759RT
PEG 8000 (40% w/v)Sigma AldrichP14584 °C
TE Buffer pH 8, 2 um filteredInvitrogenAM9858RT
Native Barcoding Kit 96 V14ONTSQK-NBD114.96See before starting notes-20 °C
NEBNext Ultra II End repair/dA-tailing ModuleNEBE7546Thaw on ice-20 °C
NEBNext FFPE Repair MixNEBM6630Thaw on ice-20 °C
NEB Blunt/TA Ligase Master MixNEBM0367Thaw on ice-20 °C
NEBNext Quick Ligation ModuleNEBE6056Thaw on ice-20 °C
Qubit Assay TubesThermoFisherQ32856RT
Qubit 1X dsDNA HS Assay KitThermoFisherQ33231Equilibrate at RT for 30 min4 °C
Genomic DNA ScreenTape Assay (optional)Agilent5067-5365; 5067-5366Equilibrate at RT for 30 min4 °C
TapeStation strips and caps (optional)Agilent401428; 401425RT
TapeStation Loading tips (optional)Agilent5067-5598RT
PromethION R10.4.1M flow cellONTFLO-PRO114MEquilibrate at RT for 20 min4 °C
Flow Cell Wash KitONTEXP-WSH004 or EXP-WSH004-XL-20 °C
General consumables:

Gloves
Pipettes: P10, P20, P200 and P1000 (any brand)
Pipette filter tips: 10 ul, 200 ul and 100 ul (any brand)
0.2 ml thin-walled PCR tubes
1.5 ml Eppendorf LoBind tubes 
2 ml Eppendorf LoBind tubes
80 % Ethanol, prepared with nuclease-free water
70 % Ethanol, prepared with nuclease-free water

Equipment:
HulaMixer (ThermoFisher) or rotating tube mixer
Magnetic rack for 0.2 ml PCR tubes
Magnetic stand for 1.5 ml Eppendorf tubes, e.g. DynaMag-2 Magnet (ThermoFisher; 12321D)
Microfuge 
Thermal cycler
Ice bucket and ice
Benchtop Cooler Rack for holding 1.5-2 ml tubes, with Temperature-20 °C   temperature range
Vortex mixer 
Qubit fluorometer (or equivalent)
Agilent TapeStation (optional)
Vortex mixer IKA MS3 with 96-well sample plate adapter (optional)
PromethION sequencer (ONT)
Heat block or thermomixer, set to 37 °C
Equipment
Magnet
NAME
DynaMag™-2 Magnet
TYPE
Thermo Fisher
BRAND
12321D
SKU
https://www.thermofisher.com/order/catalog/product/12321D
LINK
ReagentNuclease-free WaterMerck MilliporeSigma (Sigma-Aldrich)Catalog #W4502-1L 
ReagentT7 Endonuclease I - 250 unitsNew England BiolabsCatalog #M0302S   
ReagentAMPure XP BeadsBeckman CoulterCatalog #A63882  
ReagentTris-HCl pH 8.0Thermo ScientificCatalog #J22638-AE  
ReagentEDTA (0.5 M, pH 8.0, nuclease-free)Thermo Fisher ScientificCatalog #AM9260G  
Reagent5M NaCl solutionThermo Fisher ScientificCatalog #AM9759  
Reagent40% Polyethylene Glycol MW 8000Merck MilliporeSigma (Sigma-Aldrich)Catalog #P1458  
ReagentTE, pH 8.0, RNase-freeThermo FisherCatalog #AM9858  
ReagentNative barcoding kit (96)Oxford Nanopore TechnologiesCatalog #SQK-NBD114.96  
ReagentNEBNext® Ultra™ II End Repair/dA-Tailing ModuleNew England BiolabsCatalog #E7546  
ReagentNEBNext FFPE Repair MixNew England BiolabsCatalog #M6630  
ReagentNEB Blunt/TA Ligase Master Mix Contributed by usersCatalog #M0367  
ReagentNEBNext Quick Ligation ModuleNew England BiolabsCatalog #E6056S  
ReagentQubit assay tubesThermo Fisher ScientificCatalog #Q32856  
ReagentGenomic DNA ScreenTapeAgilent TechnologiesCatalog #5067-5365  
ReagentOptical tube strips (8x Strip)Agilent TechnologiesCatalog #401428  ReagentGenomic DNA ReagentsAgilent TechnologiesCatalog #5067-5366  ReagentQubit&trade; 1X dsDNA HS Assay KitThermo FisherCatalog #Q33231  
ReagentOptical tube strip caps (8x strip)Agilent TechnologiesCatalog #401425  ReagentTapeStation Loading tipsAgilent TechnologiesCatalog #5067-5598  
ReagentPromethION R10.4.1M flow cellOxford Nanopore TechnologiesCatalog #FLO-PRO114M  
ReagentONT Flow Cell Wash KitOxford Nanopore TechnologiesCatalog #EXP-WSH004  
ReagentFlow Cell Wash KitOxford Nanopore TechnologiesCatalog #EXP-WSH004-XL  

Before start

Section 1: DNA QC
Before starting:
Pre-warm Qubit 1X dsDNA HS reagent to room temp for Duration00:30:00   prior to using.
Pre-warm Genomic ScreenTape assay reagents to room temp for Duration00:30:00   prior to using.

Section 2: T7 debranching
Before starting:
Thaw 10x NEBuffer 2 TemperatureOn ice  , vortex and keep TemperatureOn ice   until needed.
Pre-warm AMPure XP beads at room temperature for Duration00:30:00   and vortex to thoroughly mix immediately prior to use.
Prepare custom AMPure buffer as follows, by adding the following and pipette mixing:

AB
ReagentVolume (ul)
1 M Tris-HCl pH 820
0.5 M EDTA pH 84
5 M NaCl640
PEG 8000 (40% w/v)550
Nuclease-free water778

Section 3: Library preparation
Before starting:
Thaw all buffers TemperatureOn ice  , vortex to mix and ensure there is no precipitate. If precipitate is visible, vortex until it has fully mixed into solution. Keep on ice until needed. Briefly spin tubes in microfuge before opening.
Keep enzymes and Native Adapter (NA) at Temperature-20 °C   until needed. Just before use, transfer enzymes to Benchtop Cooler. Mix enzymes by very gently flicking the tubes, spin tubes briefly in microfuge and keep in Benchtop Cooler.
Pre-warm AMPure XP beads at room temperature for Duration00:30:00   and vortex immediately prior to using.
Thaw NBD114.96 barcode plate (or NDB114.24 barcode tubes) at room temperature. Centrifuge plate or tubes before opening and prepare 1.25 ul aliquots of individual barcodes in 0.2 ml PCR tubes and keep on ice.
Thaw NEB Blunt/TA Ligase Master Mix on ice, mix by inversion and keep TemperatureOn ice  .
Thaw Short Fragment Buffer (SFB) at room temperature, vortex, and centrifuge briefly before using.

Section 4: Flow cell loading
Before starting:
Thaw Flow Cell Tether (FCT), Flow Cell Flush (FCF), Sequencing Buffer (SB) and Library Solution (LIS) at TemperatureRoom temperature  . Mix by vortex and centrifuge briefly before use.
Remove PromethION flow cell from Temperature4 °C   and equilibrate to TemperatureRoom temperature   for Duration00:20:00   before loading.

Section 1: DNA QC

Measure the concentration of each dMDA sample using the Qubit Fluorometer. Mix Amount1 µL   of DNA with Amount199 µL   of 1X dsDNA HS reagent, in a 0.5 ml Qubit tube. Vortex for Duration00:00:10  , spin briefly in a microfuge and incubate at TemperatureRoom temperature   for Duration00:02:00   and measure concentration.

2m 10s

(Optional) Assess the DNA integrity and fragment-length profile of each sample using the Agilent TapeStation and Genomic ScreenTape Assay. Use Amount1 µL   of DNA, at a concentration of Amount10-100 ng/µL   for TapeStation analysis.

Section 2: T7 debranching

1h 2m

Aliquot Amount500 ng  of each dMDA sample into a 0.2 ml thin-walled PCR tube and make the volume up to Amount26 µL   with nuclease-free water.

Prepare the following reaction:
AB
ReagentVolume (uL)
Nuclease-free water26-x
Template DNAx
10x NEBuffer 23
T7 endonulcease I (10 U/uL)1
Total volume30

Incubate reactions in a thermal cycler at Temperature37 °C   for Duration01:00:00  

Prepare custom SPRI beads, by replacing the buffer of AMPure XP beads with custom AMPure XP buffer (according to the ONT protocol: Ligation sequencing gDNA – whole genome amplification (SQK-LSK110) Version: WAL_9115_v110_revH_10Nov2020).

Mix AMPure XP beads by vortexing thoroughly and transfer two 1 ml aliquots into two 1.5 ml Eppendorf LoBind tubes.

Place tubes in a magnetic rack for Duration00:02:00  , to pellet the beads and then remove the supernatant.

Remove the tubes from the magnet and resuspend the beads by adding Amount1 mL   of nuclease-free water and vortexing, then return the tubes to the magnet rack.

Repeat the above wash step, for a total of 2 washes.

Briefly centrifuge the tubes, return to the magnet, and use a P20 pipette to remove any remaining water.

Resuspend and pool the two pellets in Amount200 µL   of custom AMPure buffer, then transfer the resuspended beads to the 2 ml tube containing the remaining custom AMPure buffer.

Vortex thoroughly and make sure beads are thoroughly mixed again just before use.

Adjust debranching reactions to Amount50 µL   using TE buffer (pH 8), add Amount35 µL   of custom SPRI beads and incubate on a Hula-Mixer at Centrifigation9 rpm, 00:20:00 .

20m

Pellet beads on a magnetic rack for Duration00:02:00  , or until beads are completely separated from solution and remove the supernatant.

Leave tubes on the magnet and wash the pellet by carefully adding Amount200 µL   of 70 % ethanol. Do not disturb the bead pellet and remove ethanol after Duration00:00:30  .

30s

Repeat the above wash step, for a total of two washes.

Briefly centrifuge the samples, return to the magnet, and use a P20 pipette to remove any remaining ethanol. 

Air-dry pellet for up to Duration00:02:00  .

To elute DNA from the beads, resuspend the pellet by adding Amount20 µL   of nuclease-free water and flicking the tubes to thoroughly resuspend the beads.

Briefly spin the tubes in a microfuge for 1-2 s.

Incubate in a thermal cycler at Temperature50 °C   for Duration00:01:00  .

Incubate at TemperatureRoom temperature   for Duration00:09:00  .

Pellet beads on a magnetic rack for Duration00:02:00  .

Transfer the supernatant, containing eluted DNA, to a new 0.2 ml thin-walled PCR tube.

Quantify Amount1 µL   of eluted DNA by Qubit.

Section 3: Library Preparation

56m 30s

The following library preparation steps follow the protocol Ligation sequencing gDNA – Native Barcoding Kit 24 V14; Version: NBE_9169_v114_revQ_15Sep2022, with some modifications. For each sample, use all recovered debranched dMDA DNA from step 18.

End-prep:

Prepare the following reaction for each sample, in a 0.2 ml thin-walled PCR tube:
AB
ReagentVolume (uL)
Nuclease-free water5 
Debranched dMDA sample19
NEBNext FFPE DNA Repair Buffer1.75
Ultra II End-prep Reaction Buffer1.75
Ultra II End-prep Enzyme Mix1.5
NEBNext FFPE DNA Repair Mix1
Total volume30

Mix samples by pipetting and briefly centrifuge.

Incubate reactions in a thermal cycler at Temperature20 °C   for Duration00:30:00  , then Temperature65 °C   for Duration00:05:00  .

35m

Mix AMPure XP beads by vortexing and add Amount60 µL   to each reaction.

Mix the AMPure beads with the reaction solution by thoroughly flicking and inverting the tubes.

Incubate at TemperatureRoom temperature   for Duration00:05:00  .

Briefly centrifuge the tubes then place on a magnetic rack for ~ Duration00:02:00  , or until the beads have completely pelleted.

Remove the supernatant.

Leave tubes on the magnet and wash the pellet by carefully adding Amount200 µL  of 80 % ethanol. Do not disturb the bead pellet and remove ethanol after Duration00:00:30  .

30s

Repeat the above wash step, for a total of two washes.

Briefly centrifuge the samples, return to the magnet, and use a P20 pipette to remove any remaining ethanol.

Air-dry pellet for up to Duration00:02:00  .

To elute DNA from the beads, remove tubes from the magnetic rack and resuspend the pellets by adding Amount23 µL   of nuclease-free water and flicking the tubes to thoroughly resuspend the beads. Briefly spin the tubes in a microfuge for 1-2 s and incubate in a thermal cycler at Temperature37 °C   for Duration00:10:00  .

10m

Pellet beads on a magnetic rack for Duration00:02:00   and then transfer Amount22.5 µL   of supernatant, containing eluted DNA, to a new 0.2 ml thin-walled PCR tube.

Barcode Adapter Ligation:
Prepare the following barcode adapter ligations at TemperatureRoom temperature  
AB
ReagentVolume (uL)
End-prepped DNA22.5 
Native Barcode (NB01-24)2.5
Blunt/TA Ligase Master Mix25
Total volume50

Gently pipette mix to thoroughly mix reactions and briefly centrifuge.

Incubate at TemperatureRoom temperature   for Duration00:30:00  .

30m

Mix AMPure XP beads by vortexing and add Amount100 µL   to each reaction.

Mix the AMPure beads with the reaction solution by thoroughly flicking and inverting the tubes and incubate at TemperatureRoom temperature   for Duration00:05:00  .

Briefly centrifuge the tubes then place on a magnetic rack for ~ Duration00:02:00  , or until the beads have completely pelleted.

Remove the supernatant.

Leave tubes on the magnet and wash the pellet by carefully adding Amount200 µL   of 80 % ethanol. Do not disturb the bead pellet and remove ethanol after Duration00:00:30  .

30s

Repeat the above wash step, for a total of two washes.

Briefly centrifuge the samples, return to the magnet, and use a P20 pipette to remove any remaining ethanol.

Air-dry pellet for up to Duration00:02:00  .

To elute DNA from the beads, remove tubes from the magnetic rack and resuspend the pellets by adding Amount16 µL   of nuclease-free water and flicking the tubes to thoroughly resuspend the beads. Briefly spin the tubes in a microfuge for 1-2 s and incubate in a thermal cycler at Temperature37 °C   for Duration00:10:00  .

10m

Pellet beads on a magnetic rack for Duration00:02:00   and then transfer the supernatant, containing eluted DNA, to a new 0.2 ml thin-walled PCR tube.

Quantify Amount1 µL   of eluted DNA by Qubit.

Sequencing Adapter Ligation:
Combine barcoded samples in equal amounts, in a 1.5 ml LoBind tube, and use the pooled sample to prepare the below reaction. Add reagents in the listed order:
AB
ReagentVolume (uL)
Barcoded sample pool30 
Native Adapter5
NEBNext Quick Ligation Reaction Buffer (5x)10
Quick T4 DNA Ligase5
Total volume50

Incubate ligation reaction at TemperatureRoom temperature   for Duration00:30:00  .

30m

Add Amount100 µL   of resuspended AMPure beads and incubate on a HulaMixer for Duration00:10:00   at Centrifigation9 rpm .

10m

Briefly centrifuge the tube then place on a magnetic rack for ~ Duration00:02:00  , or until the beads have completely pelleted.

Remove the supernatant.

Remove tube from the magnetic stand and wash the beads by adding Amount125 µL   of Short Fragment Buffer (SF) and thoroughly resuspend beads by flicking the tube.

Return tube to the magnetic stand and pellet beads for ~ Duration00:02:00  .

Repeat the above wash steps, for a total of two washes.

Briefly centrifuge the samples, return to the magnet, and use a P20 pipette to remove any remaining SFB.

Air-dry pellet for up to Duration00:00:30  .

30s

To elute DNA from the beads, remove the tube from the magnetic rack and resuspend the pellet by adding Amount25 µL   of Elution Buffer (EB) and flicking the tube to thoroughly resuspend the beads.

Briefly spin the tube in a microfuge for 1-2 s and incubate in a heat block or water bath at Temperature37 °C   for Duration00:10:00  .

10m

Pellet beads on a magnetic rack for Duration00:02:00   and then transfer the supernatant, containing eluted DNA, to a new 1.5 ml LoBind tube.

Quantify Amount1 µL   of eluted DNA by Qubit.

Section 4: Flow cell loading

Prepare and load PromethION flow cell according to the protocol: Ligation sequencing gDNA – Native Barcoding Kit 24 V14; Version: NBE_9169_v114_revQ_15Sep2022.

Start run in MinKNOW using default parameters for the NBD114.24 or NBD114.96 library prep kit.

Monitor number of available pores during the run and perform nuclease flush and reloads as needed, using the Flow Cell Wash Kit (EXP-WSH004 or EXP-WSH004-XL) and the protocol: Flow Cell Wash Kit (EXP-WSH004 or EXP-WSH004-XL) Version: WFC_9120_v1_revQ_08Dec2020. A Duration72:00:00   run will normally require 1-2 flush and reloads to maximise the output of the flow cell.

Protocol references

Ester Kalef-Ezra, Diego Perez-Rodriguez, Christos Proukakis 2023. Manual isolation of nuclei from human brain using CellRaft device and single nucleus Whole Genome Amplification. protocols.iohttps://dx.doi.org/10.17504/protocols.io.kxygxzjjov8j/v1

A	B	C	D	E
Item	Supplier	Catalogue Number	Preparation prior to use	Storage
Nuclease-free water	Sigma Aldrich	W4502	Aliquot	RT
T7 Endonuclease I (10U/ul)	NEB	M0302S	Place on ice	-20 °C
10X NEBuffer 2	NEB	M0302S	Thaw on ice	-20 °C
AMPure XP Beads	Beckman Coulter	A63882	Equilibrate at RT for 30 min	4 °C
1 M Tris-HCl pH 8	Thermo Scientific	J22638-AE		RT
0.5 M EDTA pH 8, nuclease-free	Thermo Fisher Scientific	AM9260G		RT
5M NaCl	Thermo Fisher Scientific	AM9759		RT
PEG 8000 (40% w/v)	Sigma Aldrich	P1458		4 °C
TE Buffer pH 8, 2 um filtered	Invitrogen	AM9858		RT
Native Barcoding Kit 96 V14	ONT	SQK-NBD114.96	See before starting notes	-20 °C
NEBNext Ultra II End repair/dA-tailing Module	NEB	E7546	Thaw on ice	-20 °C
NEBNext FFPE Repair Mix	NEB	M6630	Thaw on ice	-20 °C
NEB Blunt/TA Ligase Master Mix	NEB	M0367	Thaw on ice	-20 °C
NEBNext Quick Ligation Module	NEB	E6056	Thaw on ice	-20 °C
Qubit Assay Tubes	ThermoFisher	Q32856		RT
Qubit 1X dsDNA HS Assay Kit	ThermoFisher	Q33231	Equilibrate at RT for 30 min	4 °C
Genomic DNA ScreenTape Assay (optional)	Agilent	5067-5365; 5067-5366	Equilibrate at RT for 30 min	4 °C
TapeStation strips and caps (optional)	Agilent	401428; 401425		RT
TapeStation Loading tips (optional)	Agilent	5067-5598		RT
PromethION R10.4.1M flow cell	ONT	FLO-PRO114M	Equilibrate at RT for 20 min	4 °C
Flow Cell Wash Kit	ONT	EXP-WSH004 or EXP-WSH004-XL		-20 °C

	A	B
	Reagent	Volume (ul)
	1 M Tris-HCl pH 8	20
	0.5 M EDTA pH 8	4
	5 M NaCl	640
	PEG 8000 (40% w/v)	550
	Nuclease-free water	778

	A	B
	Reagent	Volume (uL)
	End-prepped DNA	22.5
	Native Barcode (NB01-24)	2.5
	Blunt/TA Ligase Master Mix	25
	Total volume	50

	A	B
	Reagent	Volume (uL)
	Barcoded sample pool	30
	Native Adapter	5
	NEBNext Quick Ligation Reaction Buffer (5x)	10
	Quick T4 DNA Ligase	5
	Total volume	50

Public workspaceOxford Nanopore Technologies (ONT) library preparation and sequencing of DNA prepared using droplet Multiple Displacement Amplification (dMDA)

Oxford Nanopore Technologies (ONT) library preparation and sequencing of DNA prepared using droplet Multiple Displacement Amplification (dMDA)