TITLE: Cost-reducing Nanopore Library Preparation for R10 Native Barcoding Kit
AUTHORS: Guan Jie Phang

[DESCRIPTION]
Cost-reducing protocols for Nanopore R10 flow cell Native Barcoding library construction.

[STEPS]
SECTION: Step1. End-prep
2.  In 200 µL tube(s), combine the following chemicals per tube:
 

 


 

SECTION: Step1. End-prep
2.1. Incubate in a thermocycler at 20 °C for 30 min and 65 °C for 30 min. Hold at 4 °C 
SECTION: Step2. Adapter ligation
21. Thaw the Native Adapter (LA) and KAPA HyperPrep Ligation Buffer. Vortex and spin down the tubes, and place on ice immediately afterwards.
 

 


SECTION: Step2. Adapter ligation
21.2. In a 1.5 mL tube, add the following chemicals:
 

 

SECTION: Step2. Adapter ligation
21.3. Incubate overnight at 20 Room temperature .

SECTION: Step3. Cleanup
27. Wash the beads by adding either 125 µL Long Fragment Buffer (LFB).  Flick the beads to resuspend, spin down, then return the tube to the magnetic rack and allow the beads to pellet.

 

SECTION: Step3. Cleanup
23. Resuspend the AMPure XP beads by vortexing.
SECTION: Step3. Cleanup
27.1. Remove the supernatant using a pipette and discard.
SECTION: Step4. Priming and loading the SpotON flow cell
33. Thaw the flow cell, Sequencing Buffer (SQB), Loading Solution (LIS), and one tube of Flush mixture (FB & FLT) at room temperature on cooling block.

 

SECTION: Step5. Flow Cell Wash and Storage
45. Place the tube of Wash Mix (WMX) on ice. Do not vortex the tube.
Thaw one tube of Wash Diluent (DIL) at room temperature on a cooling block.
 

SECTION: Step5. Flow Cell Wash and Storage
45.1. In a clean 1.5 mL microtube, prepare the following Flow Cell Wash Mix:
 

 

SECTION: Step5. Flow Cell Wash and Storage
45.2. Lift the sensory cover.

 

SECTION: Step5. Flow Cell Wash and Storage
45.3. Rotate the flow cell priming port cover clockwise so that the priming port is visible.
SECTION: Step5. Flow Cell Wash and Storage
45.4. After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles:
1. Set a P1000 pipette to 800 μl.
2. Insert the tip into the priming port.
3. Turn the wheel until the dial shows 820–830 μl, or until you can see a small volume of liquid entering the pipette tip.
 

SECTION: Step5. Flow Cell Wash and Storage
45.5. Load 200 μl of the prepared Flow Cell Wash Mix into the flow cell via the priming port, avoiding the introduction of air.
SECTION: Step5. Flow Cell Wash and Storage
45.6. Close the priming port and keep into 4°C for 2 days.
SECTION: Step3. Cleanup
22. Thaw either Long Fragment Buffer (LFB) or Short Fragment Buffer (SFB) at room temperature on a cooling block, mix by vortexing, spin down and place on ice.
SECTION: Step3. Cleanup
26. Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.

 

SECTION: Step3. Cleanup
25. Incubate for 10 minutes at Room temperature.
SECTION: Step3. Cleanup
24. Add 0.45X AMPure XP Beads (AXP) to the tube and mix by flicking the tube.

SECTION: Step3. Cleanup
29. Spin down and place the tube back on the magnet. Pipette off any residual supernatant. Allow to dry for 5~30 seconds.
 

SECTION: Step3. Cleanup
30. Remove the tube from the magnetic rack and resuspend the pellet in 15 µL Elution Buffer (EB). Spin down and incubate for 10 minutes at 37 °C.

SECTION: Step3. Cleanup
28.  . 
SECTION: Step3. Cleanup
31. Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
SECTION: Step3. Cleanup
32. Leave the tube aside, and go to the priming steps.

 

SECTION: Step2. Adapter ligation
21.1. Spin down the KAPA HyperPrep Ligase and place on ice immediately.

SECTION: Step4. Priming and loading the SpotON flow cell
34. Open the MinION device lid and slide the flow cell under the clip. Press down firmly on the flow cell to ensure correct thermal and electrical contact.
 

 

SECTION: Step4. Priming and loading the SpotON flow cell
35. Slide the flow cell priming port cover clockwise to open the priming port.
 

SECTION: Step4. Priming and loading the SpotON flow cell
36. After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles: 
1. Set a P1000 pipette to 800 μl.
2. Insert the tip into the priming port.
3. Turn the wheel until the dial shows 820–230 µl, or until you can see a small volume of buffer entering the pipette tip.
 

SECTION: Step4. Priming and loading the SpotON flow cell
37. Load 800 μl of the priming mix into the flow cell via the priming port by turning the pipet wheel, avoiding the introduction of air bubbles. Wait for 5 minutes. During this time, prepare the library for loading by following the steps below.
SECTION: Step4. Priming and loading the SpotON flow cell
39. Complete the flow cell priming:
1. Gently lift the SpotON sample port cover to make the SpotON sample port accessible.
2. Load 200 μl of the priming mix into the flow cell via the priming port (not the SpotON sample port), avoiding the introduction of air bubbles.
 

SECTION: Step4. Priming and loading the SpotON flow cell
38. Aspirate 14 µL of the eluted prepared library into a new 1.5 mL microtube.
SECTION: Step4. Priming and loading the SpotON flow cell
38.1. In that new tube, prepare the library for loading as follows:
 

 

SECTION: Step4. Priming and loading the SpotON flow cell
40. Mix the prepared library gently by pipetting up and down in the tip using a P200 pipet just prior to loading.
SECTION: Step4. Priming and loading the SpotON flow cell
41. Add the library to the flow cell via the SpotON sample port in a dropwise fashion. Ensure each drop flows into the port before adding the next.
SECTION: Step4. Priming and loading the SpotON flow cell
42. Gently close the SpotON sample port cover, making sure the bung enters the SpotON port, close the priming port, cover the sensor array with the sensory cover, and close the MinION device lid.
 

SECTION: Step5. Flow Cell Wash and Storage
44. Stop or pause the sequencing experiment in MinKNOW, and leave the flow cell in the device.
 

SECTION: Step5. Flow Cell Wash and Storage
45.7.   

SECTION: Step5. Flow Cell Wash and Storage
47. Thaw one tube of Storage Buffer (S) at room temperature on a cooling block.
SECTION: Step5. Flow Cell Wash and Storage
47.1. Mix contents thoroughly by pipetting and spin down briefly.
SECTION: Step5. Flow Cell Wash and Storage
46. Take out the MinION device from 4°C fridge.
SECTION: Step5. Flow Cell Wash and Storage
48. Rotate the flow cell priming port cover clockwise so that the priming port is visible. Open the device lid.
SECTION: Step5. Flow Cell Wash and Storage
49. After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles:
1. Set a P1000 pipette to 800 μl.
2. Insert the tip into the priming port.
3. Turn the wheel until the dial shows 820–830 μl, or until you can see a small volume of liquid entering the pipette tip.
 

SECTION: Step5. Flow Cell Wash and Storage
50. Add 500 μl of Storage Buffer (S) through the priming port of the flow cell by turning the pipet wheel.
SECTION: Step5. Flow Cell Wash and Storage
50.1. Close the priming port.
SECTION: Step5. Flow Cell Wash and Storage
51. Using a P1000, remove all fluids from the waste channel through Waste port 2. As both the priming port and SpotON sample port are closed, no fluid should leave the sensor array area.

 

 

SECTION: Step5. Flow Cell Wash and Storage
52. Perform a Flow Cell Check.
SECTION: Step5. Flow Cell Wash and Storage
53. The flow cell can now be stored at 4°C.
SECTION: Step3. Native barcode ligation
3. Thaw the Native Barcodes (NB) required for your number of samples at room temperature on a cooling block.
 

SECTION: Step3. Native barcode ligation
4. In a clean 200 µL tube, add the reagents in the following order per tube:
 
 
 


SECTION: Step3. Native barcode ligation
5. Incubate for 20 minutes at Room temperature. 

SECTION: Step3. Native barcode ligation
7. Add 2 µL of EDTA to each tube and mix thoroughly by flicking the tube and spin down brieftly.
SECTION: Step3. Native barcode ligation
6. Thaw EDTA at Room temperature on a cooling block, mix by vortexing, spin down, and place on ice.

SECTION: Step3. Native barcode ligation
8. Pool all the barcoded samples in a 1.5 mL microtube.
 

SECTION: Step3. Native barcode ligation
10. Resuspend the AMPure XP beads by vortexing.
SECTION: Step3. Native barcode ligation
11. Add 0.45X AMPure XP Beads (AXP) to the tube and mix by flicking the tube.

SECTION: Step3. Native barcode ligation
12. Incubate for 10 minutes at Room temperature.
SECTION: Step3. Native barcode ligation
13. Spin down the sample and pellet on a magnet for 5 mintues. Keep the tube on the magnet until the elute is clear and colorless, and pipette off the supernatant.

SECTION: Step3. Native barcode ligation
14. Keep the tube on the magnetic rack and wash with 700 µL of freshly prepared 80% ethanol without disturbing the pellet. Flip the magnetic rack for 30 seconds. Remove the ethanol using a pipet and discard.

SECTION: Step3. Native barcode ligation
15.  . 
SECTION: Step3. Native barcode ligation
16. Spin down and place the tube back on the magnet. Pipette off any residual supernatant. Allow to dry for 5~30 seconds.
 

SECTION: Step3. Native barcode ligation
17. Remove the tube from the magnetic rack and resuspend the pellet in 35 µL ddH2O by gently flicking.
SECTION: Step3. Native barcode ligation
19. Pellet the beads on a magnet until the eluate is clear and colourless.
SECTION: Step3. Native barcode ligation
9. Prepare fresh 80% ethanol in ddH2O.
SECTION: Step3. Native barcode ligation
18. Incubate for 10 minutes at 37 °C.
SECTION: Step3. Native barcode ligation
20. Remove and retain 35 µL of elute into a clean 1.5 mL microtube.
SECTION: Step4. Priming and loading the SpotON flow cell
43. Quantify 1 µL of pooled barcoded sample and eluted sample using Qubit.
SECTION: Step1. End-prep
1. Make sure you have high concentration of DNA (>100 ng/µL, quantified by Qubit).