TITLE: Sequencing dMDA Products on the MinION using Oxford Nanopore&#39;s Rapid Barcoding Kit
AUTHORS: Dominic Horner, Ester Kalef-Ezra, Marco Toffoli, Christos Proukakis

[DESCRIPTION]
Using Oxford Nanopore MinION and the Rapid Barcoding library preparation kit, we perform long read sequencing on droplet multiple displacement amplification (dMDA) products generated from single nuclei. Single nuclei contain around 6.6pg of DNA, to sequence this miniscule amount of DNA using long reads a whole genome amplification (WGA) method must be used to provide enough starting material for library preparation. MDA is currently the only WGA method which produces reads >10kb in length. Sequencing DNA of this length using long reads can allow for direct resolution and identification of most structural variants, including large LINE-1 insertions and deletions. dMDA is an improvement of the MDA technique, partitioning DNA template molecules into droplets prevents the generation of intermolecular chimeras, and a finite amount of dMDA reagents being packed into each droplet, prevents overamplification of certain reads.
The expected result of this protocol is to be able to produce and sequence libraries generated from dMDA products.

[STEPS]
SECTION: Prepare Samples and Reagents
1. 
Remove Ampure XP beads from fridge and allow them to equilibrate to Room temperature  for approximately 30 min. Mix well by vortexing prior to use, ensure beads are resuspended and appear homogenous.

SECTION: dMDA product purification
5. 
Add Ampure XP beads in a 0.8X ratio by volume to each sample you wish to purify.

For example to purify 10 µL  of sample add 8 µL  of Beads.
SECTION: Rapid barcoding PCR
20. 
Thaw the following components of the Rapid PCR Barcoding and sequencing kit (SQK-RBP004)
on ice :

Fragmentation Mix (FRM)
Rapid Adapter (RAP)

and thaw the Barcodes (RLB 01-12A) you intend to use at Room temperature for 30 min .


SECTION: Rapid barcoding PCR
21. 
Per sample, in fresh, labelled 0.2 ml tubes transfer 1-5 ng of sample  in 3 µL  of nuclease free water. Mix gently by flicking the side of the tube and spin down briefly in a microcentrifuge.


 


SECTION: Rapid barcoding PCR
22. 
Add 1 µL  of Fragmentation Mix to each 3 µL  sample, mix gently by flicking and spin down in a microcentrifuge.
SECTION: Rapid barcoding PCR
23.   
Transfer all tubes to a thermal cycle and incubate at 30 °C  for 1 min then 80 °C for 1 min . Once finished briefly place tubes on ice  to cool down.

SECTION: Rapid barcoding PCR
24. 
To each PCR tube containing 4 µL  of tagmented DNA add20 µL of nuclease free water, 1 µL of desired barcode (RLB 01-12A, at10 μM ) and 25 µL of LongAmp Taq 2x master mix.

SECTION: Rapid barcoding PCR
25. 
Mix gently by flicking the tubes and then spin briefly down in microcentrifuge.
SECTION: Rapid barcoding PCR
26. 
Transfer samples to a thermal cycler and begin barcoding PCR using the cycling conditions in the table below:

 

SECTION: PCR cleanup
27. 
Transfer each sample to fresh, labelled 1.5 ml Eppendorf DNA LoBind tubes.
SECTION: PCR cleanup
29. 
Resuspend Ampure XP Beads, vortex until beads are resuspended and appear homogenous.
SECTION: PCR cleanup
30. 
Add 30 µL of Ampure beads to each reaction and pipette mix 15 times using 70% total reaction volume. 

SECTION: PCR cleanup
31. 
Incubate samples on a Hula mixer for  5 min at Room temperature .

SECTION: PCR cleanup
28. 
Prepare a fresh solution of 70 - 80% Ethanol, a minimum volume of 400 µL  per sample is required for PCR clean-up.
SECTION: PCR cleanup
32. 
Remove samples from the Hula mixer and briefly spin down in a microcentrifuge.
SECTION: PCR cleanup
33. 
Place samples on a magnetic rack and allow beads to pellet for 5 min or until supernatant is clear. Then, keeping the tubes on the magnetic rack pipette off and discard supernatant.

SECTION: PCR cleanup
34. 
Keeping the tubes containing the beads on the magnetic rack, add 200 µL  of 70 - 80% Ethanol to each and then immediately pipette off and discard. Be careful not to disturb the bead pellets.

SECTION: PCR cleanup
35. 
Repeat step 34.
SECTION: PCR cleanup
36. 
Remove tubes from magnetic rack and briefly spin down in microcentrifuge.    
SECTION: PCR cleanup
37. 
 Return tubes to the magnetic rack and remove excess ethanol from each tube using a P10 pipette.

 

SECTION: PCR cleanup
38. 
Allow beads to dry for around 30 s. Do not dry beads to point of cracking.

SECTION: PCR cleanup
39. 
Remove tubes from magnetic rack and resuspend in 11 µL of 10 mM  Tris-HCl pH 8 with 50 mM  NaCl. Incubate for 2 min at Room temperature .

SECTION: PCR cleanup
40. 
Return tubes to the magnetic rack and allow beads to pellet for 5 min or until the supernatant is clear.

SECTION: PCR cleanup
41. 
For each sample, transfer 10 µL  of eluate into labelled, fresh 1.5 ml tubes and dispose of pelleted beads. 

SECTION: PCR cleanup
42. 
Quantify 1 µL  of each purified Library using the Qubit dsDNA HS assay kit.

SECTION: Library pooling, flow cell loading, and sequencing
43. 
Dilute and pool samples as necessary to generate a final pool of barcoded libraries, with a molarity between 50-100 fmol  in 10 µL  of 10 mM Tris-HCl pH 8 with 50 mM  NaCl. 


 
    

SECTION: Library pooling, flow cell loading, and sequencing
44. 
Add 1 µL  of Rapid Adaptor Protein (RAP) to the pool.


 


SECTION: Library pooling, flow cell loading, and sequencing
45. 
Mix the tube gently by flicking and spin down briefly using a microcentrifuge.
SECTION: Library pooling, flow cell loading, and sequencing
46. 
Incubate the reaction for 5 min at Room temperature .


 


SECTION: Library pooling, flow cell loading, and sequencing
47. 
Thaw sequencing buffer (SQB), loading beads (LB), flush tether (FLT), and one tube of flush buffer (FB) at Room temperature . Mix reagents by vortexing and spin down using a microcentrifuge.

SECTION: Library pooling, flow cell loading, and sequencing
48. 
Prepare a flow cell priming mix by adding 30 µL of flush tether directly to a tube of thawed and mixed flush buffer, then mix by vortexing.

SECTION: Prepare Samples and Reagents
2. 
Prepare dMDA product(s) for bead purification in 0.2ml PCR tubes. 

        
 

SECTION: Prepare Samples and Reagents
3. 
Prepare a fresh solution of 70 - 80% ethanol using nuclease free water and molecular grade ethanol. 

A minimum volume of 800 µL  of 70 - 80% ethanol is required per sample for this protocol.
SECTION: Prepare Samples and Reagents
4. 
Perform a flow cell quality check the day before or on the same day as sequencing. Ensure the pore scan recognises a minimum of 800 pores remaining for sequencing.


 

SECTION: Library pooling, flow cell loading, and sequencing
49. 
Open the MinION device and slide the flow cell under the clip.
SECTION: Library pooling, flow cell loading, and sequencing
50. 
Open the priming port by sliding it 90° Clockwise.
SECTION: Library pooling, flow cell loading, and sequencing
51. 
Take a P1000 pipette, set it to 200 µL and gently press the pipette tip into the priming port. 
Without pressing down on the pipette, slowly alter the volume to 220-230 µL. Stop turning when you notice a small amount of liquid enter the pipette tip. 
 


SECTION: Library pooling, flow cell loading, and sequencing
52. 
Load 800 µL of priming mix into the flow cell priming port. Adopting a similar method to before, this time place the pipette tip into the priming port and slowly reduce the volume, until almost all of the priming mix in the tip has entered the priming port. To avoid introducing bubbles leave the last 20-40 µL in the tip.

SECTION: Library pooling, flow cell loading, and sequencing
53. 
Wait for 5 min and during this time prepare the pool for loading.

SECTION: Library pooling, flow cell loading, and sequencing
54. 
To prepare the pool for loading, in a new tube add 30 µL of Sequencing Buffer (SQB),25.5 µL  of Loading Beads (LB), mix immediately before use, 4.5 µL of nuclease free water, and 11 µL of the DNA sequencing pool + RAP.
 


SECTION: Library pooling, flow cell loading, and sequencing
55. 
Gently lift the SpotON port cover making the SpotON sample port accessible.
SECTION: Library pooling, flow cell loading, and sequencing
56. 
Load 200 µL of priming mix into the flow cell PRIMING port, by gently pressing the tip of a P1000 pipette into the port and slowly reducing the pipette volume until 20-30 µL  remains in the tip, to avoid introducing bubbles.
 

SECTION: Library pooling, flow cell loading, and sequencing
57. 
Mix the prepared pool gently, pipette up and down 15 times using the full 75 µL volume immediately prior to loading, to ensure beads are well mixed with sample and buffer.

SECTION: Library pooling, flow cell loading, and sequencing
58. 
Add75 µL of pool to the SpotON sample port in a drop-wise fashion. Allow each droplet to be pulled into the flow cell before adding the next.
 


SECTION: Library pooling, flow cell loading, and sequencing
59. 
Gently return the SpotON sample port cover, ensure the bung enters the hole. Close the priming port and replace the MinION device lid.
SECTION: Library pooling, flow cell loading, and sequencing
60. 
Take the MinION device to a computer loaded with MinKNOW and setup experiment according to Oxford Nanopore's guidelines.
SECTION: dMDA product purification
6. 
Pipette mix 20 times using 70% of total reaction volume.
SECTION: dMDA product purification
7. 
Briefly spin-down using micro centrifuge and incubate at room temperature for 5 min .
SECTION: dMDA product purification
8. 
Place each sample onto a magnetic rack and incubate for 5 min , or until supernatant is clear.
SECTION: dMDA product purification
9. 
Pipette off and discard supernatant, avoid touching the bead pellet.
SECTION: dMDA product purification
10. 
Add 200 µL  of 80% ethanol to each tube, then immediately pipette off.

If available, use a reagent reservoir and a P200 multi-channel pipette when working with multiple samples.
SECTION: dMDA product purification
11. 
Repeat step 10.
SECTION: dMDA product purification
12. 
Remove samples from magnetic rack and briefly spin down bead-pellet on a micro centrifuge, then return to magnetic rack and use a P10 pipette to remove any excess ethanol. 


 

SECTION: dMDA product purification
13. 
Leave tube lids open and allow beads to dry on Magnetic rack for around 30 s .

Be careful not to over-dry the beads as this may make it difficult to get them back to go back into solution during the following step.
SECTION: dMDA product purification
14. 
Remove samples from the rack and resuspend beads in 11-21 µL of Nuclease free water. Mix well by pipetting 15 times at 70% total reaction volume, or until beads are completely resuspended.
SECTION: dMDA product purification
15. 
Incubate at Room temperature  for 5 min .
SECTION: dMDA product purification
16. 
Return samples to the magnetic rack and allow beads to separate for 2 min or until supernatant becomes is clear.
SECTION: dMDA product purification
17. 
Transfer 10-20 µL of supernatant to a fresh, labelled, 0.2 ml PCR tube and prepare for Qubit quantification.

SECTION: dMDA product purification
18. 
Quantify 1 µL of sample using dsDNA HS qubit assay, following manufacturers protocol.
SECTION: dMDA product purification
19. 
Record concentration and proceed to "Perform Rapid PCR Barcoding".

Alternatively, store samples at 4 °C     or for longer term storage store at -20 °C .