TITLE: Oxford Nanopore Technologies (ONT) library preparation and sequencing of DNA prepared using droplet Multiple Displacement Amplification (dMDA)
AUTHORS: Nadine Holmes, Ester Kalef-Ezra, Christos Proukakis

[DESCRIPTION]
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.

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

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

 

Section 3: Library preparation
Before starting:
Thaw all buffers on 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 -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 30 min 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 on 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 Room temperature. Mix by vortex and centrifuge briefly before use.
Remove PromethION flow cell from 4 °C and equilibrate to Room temperature for 20 min before loading.

[STEPS]
SECTION: Section 1: DNA QC
1. 
Measure the concentration of each dMDA sample using the Qubit Fluorometer. Mix 1 µL of DNA with 199 µL of 1X dsDNA HS reagent, in a 0.5 ml Qubit tube. Vortex for 10 s, spin briefly in a microfuge and incubate at Room temperature for 2 min and measure concentration.

SECTION: Section 1: DNA QC
2. 
(Optional) Assess the DNA integrity and fragment-length profile of each sample using the Agilent TapeStation and Genomic ScreenTape Assay. Use 1 µL of DNA, at a concentration of 10-100 ng/µL for TapeStation analysis.

SECTION: Section 2: T7 debranching
3. 
Aliquot 500 ngof each dMDA sample into a 0.2 ml thin-walled PCR tube and make the volume up to 26 µL with nuclease-free water.

SECTION: Section 2: T7 debranching
4. 
Prepare the following reaction:
 

SECTION: Section 2: T7 debranching
5. 
Incubate reactions in a thermal cycler at 37 °C for 60 min

SECTION: Section 2: T7 debranching
6. 
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).
SECTION: Section 2: T7 debranching
6.1. 
Mix AMPure XP beads by vortexing thoroughly and transfer two 1 ml aliquots into two 1.5 ml Eppendorf LoBind tubes.
SECTION: Section 2: T7 debranching
6.2. 
Place tubes in a magnetic rack for 2 min, to pellet the beads and then remove the supernatant.

SECTION: Section 2: T7 debranching
6.3. 
Remove the tubes from the magnet and resuspend the beads by adding 1 mL of nuclease-free water and vortexing, then return the tubes to the magnet rack.

SECTION: Section 2: T7 debranching
6.4. 
Repeat the above wash step, for a total of 2 washes.
SECTION: Section 2: T7 debranching
6.5. 
Briefly centrifuge the tubes, return to the magnet, and use a P20 pipette to remove any remaining water.
SECTION: Section 2: T7 debranching
6.6. 
Resuspend and pool the two pellets in 200 µL of custom AMPure buffer, then transfer the resuspended beads to the 2 ml tube containing the remaining custom AMPure buffer.

SECTION: Section 2: T7 debranching
6.7. 
Vortex thoroughly and make sure beads are thoroughly mixed again just before use.
SECTION: Section 2: T7 debranching
7. 
Adjust debranching reactions to 50 µL using TE buffer (pH 8), add 35 µL of custom SPRI beads and incubate on a Hula-Mixer at 9 rpm, 20 min.

SECTION: Section 2: T7 debranching
8. 
Pellet beads on a magnetic rack for 2 min, or until beads are completely separated from solution and remove the supernatant.

SECTION: Section 2: T7 debranching
9. 
Leave tubes on the magnet and wash the pellet by carefully adding 200 µL of 70 % ethanol. Do not disturb the bead pellet and remove ethanol after 30 s.

SECTION: Section 2: T7 debranching
10. 
Repeat the above wash step, for a total of two washes.
SECTION: Section 2: T7 debranching
11. 
Briefly centrifuge the samples, return to the magnet, and use a P20 pipette to remove any remaining ethanol. 
SECTION: Section 2: T7 debranching
12. 
Air-dry pellet for up to 2 min.

SECTION: Section 2: T7 debranching
13. 
To elute DNA from the beads, resuspend the pellet by adding 20 µL of nuclease-free water and flicking the tubes to thoroughly resuspend the beads.

SECTION: Section 2: T7 debranching
14. 
Briefly spin the tubes in a microfuge for 1-2 s.
SECTION: Section 2: T7 debranching
15. 
Incubate in a thermal cycler at 50 °C for 1 min.

SECTION: Section 2: T7 debranching
16. 
Incubate at Room temperature for 9 min.

SECTION: Section 2: T7 debranching
17. 
Pellet beads on a magnetic rack for 2 min.

SECTION: Section 2: T7 debranching
18. 
Transfer the supernatant, containing eluted DNA, to a new 0.2 ml thin-walled PCR tube.

SECTION: Section 2: T7 debranching
19. 
Quantify 1 µL of eluted DNA by Qubit.

SECTION: Section 3: Library Preparation
20. 
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:
 

SECTION: Section 3: Library Preparation
21. 
Mix samples by pipetting and briefly centrifuge.

SECTION: Section 3: Library Preparation
22. 
Incubate reactions in a thermal cycler at 20 °C for 30 min, then 65 °C for 5 min.

SECTION: Section 3: Library Preparation
23. 
Mix AMPure XP beads by vortexing and add 60 µL to each reaction.

SECTION: Section 3: Library Preparation
24. 
Mix the AMPure beads with the reaction solution by thoroughly flicking and inverting the tubes.

SECTION: Section 3: Library Preparation
25. 
Incubate at Room temperature for 5 min.

SECTION: Section 3: Library Preparation
26. 
Briefly centrifuge the tubes then place on a magnetic rack for ~ 2 min, or until the beads have completely pelleted.

SECTION: Section 3: Library Preparation
27. 
Remove the supernatant.

SECTION: Section 3: Library Preparation
28. 
Leave tubes on the magnet and wash the pellet by carefully adding 200 µLof 80 % ethanol. Do not disturb the bead pellet and remove ethanol after 30 s.

SECTION: Section 3: Library Preparation
29. 
Repeat the above wash step, for a total of two washes.

SECTION: Section 3: Library Preparation
30. 
Briefly centrifuge the samples, return to the magnet, and use a P20 pipette to remove any remaining ethanol.

SECTION: Section 3: Library Preparation
31. 
Air-dry pellet for up to 2 min.

SECTION: Section 3: Library Preparation
32. 
To elute DNA from the beads, remove tubes from the magnetic rack and resuspend the pellets by adding 23 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 37 °C for 10 min.

SECTION: Section 3: Library Preparation
33. 
Pellet beads on a magnetic rack for 2 min and then transfer 22.5 µL of supernatant, containing eluted DNA, to a new 0.2 ml thin-walled PCR tube.

SECTION: Section 3: Library Preparation
34. Barcode Adapter Ligation:
Prepare the following barcode adapter ligations at Room temperature
 


SECTION: Section 3: Library Preparation
35. 
Gently pipette mix to thoroughly mix reactions and briefly centrifuge.

SECTION: Section 3: Library Preparation
36. 
Incubate at Room temperature for 30 min.

SECTION: Section 3: Library Preparation
37. 
Mix AMPure XP beads by vortexing and add 100 µL to each reaction.

SECTION: Section 3: Library Preparation
38. 
Mix the AMPure beads with the reaction solution by thoroughly flicking and inverting the tubes and incubate at Room temperature for 5 min.

SECTION: Section 3: Library Preparation
39. 
Briefly centrifuge the tubes then place on a magnetic rack for ~ 2 min, or until the beads have completely pelleted.

SECTION: Section 3: Library Preparation
40. 
Remove the supernatant.


SECTION: Section 3: Library Preparation
41. 
Leave tubes on the magnet and wash the pellet by carefully adding 200 µL of 80 % ethanol. Do not disturb the bead pellet and remove ethanol after 30 s.

SECTION: Section 3: Library Preparation
42. 
Repeat the above wash step, for a total of two washes.

SECTION: Section 3: Library Preparation
43. 
Briefly centrifuge the samples, return to the magnet, and use a P20 pipette to remove any remaining ethanol.

SECTION: Section 3: Library Preparation
44. 
Air-dry pellet for up to 2 min.

SECTION: Section 3: Library Preparation
45. 
To elute DNA from the beads, remove tubes from the magnetic rack and resuspend the pellets by adding 16 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 37 °C for 10 min.

SECTION: Section 3: Library Preparation
46. 
Pellet beads on a magnetic rack for 2 min and then transfer the supernatant, containing eluted DNA, to a new 0.2 ml thin-walled PCR tube.

SECTION: Section 3: Library Preparation
47. Quantify 1 µL of eluted DNA by Qubit.

SECTION: Section 3: Library Preparation
48. 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:
 

SECTION: Section 3: Library Preparation
49. 
Incubate ligation reaction at Room temperature for 30 min.

SECTION: Section 3: Library Preparation
50. 
Add 100 µL of resuspended AMPure beads and incubate on a HulaMixer for 10 min at 9 rpm.

SECTION: Section 3: Library Preparation
51. 
Briefly centrifuge the tube then place on a magnetic rack for ~ 2 min, or until the beads have completely pelleted.

SECTION: Section 3: Library Preparation
52. 
Remove the supernatant.

SECTION: Section 3: Library Preparation
53. 
Remove tube from the magnetic stand and wash the beads by adding 125 µL of Short Fragment Buffer (SF) and thoroughly resuspend beads by flicking the tube.

SECTION: Section 3: Library Preparation
54. 
Return tube to the magnetic stand and pellet beads for ~ 2 min.

SECTION: Section 3: Library Preparation
55. 
Repeat the above wash steps, for a total of two washes.

SECTION: Section 3: Library Preparation
56. 
Briefly centrifuge the samples, return to the magnet, and use a P20 pipette to remove any remaining SFB.

SECTION: Section 3: Library Preparation
57. 
Air-dry pellet for up to 30 s.

SECTION: Section 3: Library Preparation
58. 
To elute DNA from the beads, remove the tube from the magnetic rack and resuspend the pellet by adding 25 µL of Elution Buffer (EB) and flicking the tube to thoroughly resuspend the beads.

SECTION: Section 3: Library Preparation
59. 
Briefly spin the tube in a microfuge for 1-2 s and incubate in a heat block or water bath at 37 °C for 10 min.

SECTION: Section 3: Library Preparation
60. 
Pellet beads on a magnetic rack for 2 min and then transfer the supernatant, containing eluted DNA, to a new 1.5 ml LoBind tube.

SECTION: Section 3: Library Preparation
61. 
Quantify 1 µL of eluted DNA by Qubit.

SECTION: Section 4: Flow cell loading
62. 
Prepare and load PromethION flow cell according to the protocol: Ligation sequencing gDNA – Native Barcoding Kit 24 V14; Version: NBE_9169_v114_revQ_15Sep2022.

SECTION: Section 4: Flow cell loading
63. 
Start run in MinKNOW using default parameters for the NBD114.24 or NBD114.96 library prep kit.

SECTION: Section 4: Flow cell loading
64. 
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 4320 min run will normally require 1-2 flush and reloads to maximise the output of the flow cell.