TITLE: Nanopore amplicon sequencing
AUTHORS: Yoshiyuki  Matsuo

[DESCRIPTION]
The two-step PCR method allows us to perform nanopore amplicon sequencing with a user-defined inner primer set combined with barcoded outer primers provided by Oxford Nanopore Technologies, taking advantage of rapid adapter attachment chemistry. This method can be applied to a wide range of sequence-based analyses, including microbiome profiling and the identification of genetic variations in targeted loci.

[STEPS]
SECTION: Workflow
1.   

SECTION: 1st PCR with inner primers
2. Prepare the PCR master mix.
 

Inner primers (user-supplied)
 

 

SECTION: 1st PCR with inner primers
3. Perform PCR.
 

 

SECTION: 1st PCR with inner primers
4. Analyze 2 µL of the PCR products by gel electrophoresis to verify successful amplification.

 

 

SECTION: 2nd PCR with barcoded outer primers
5. Prepare the PCR master mix.
 

 

SECTION: 2nd PCR with barcoded outer primers
6. Perform PCR.
 

 

SECTION: 2nd PCR with barcoded outer primers
7. Analyze 1 µL of the PCR products by gel electrophoresis.

 

 

SECTION: PCR cleanup
8. Resuspend the AMPure XP beads by vortexing.
SECTION: PCR cleanup
9. Add AMPure XP beads to the sample and mix by pipetting.
 

 

SECTION: PCR cleanup
10. Incubate at Room temperature for 5 min.

SECTION: PCR cleanup
11. Place the tube on a magnetic rack for 2 min.

 

SECTION: PCR cleanup
12. Pipette off the supernatant.
SECTION: PCR cleanup
13. Wash the beads with 70% ethanol as follows (1/2).


SECTION: PCR cleanup
15. Spin down and place the tube back in the magnetic rack.
SECTION: PCR cleanup
16. Pipette off any residual ethanol.
SECTION: PCR cleanup
17. Remove the tube from the magnetic rack and resuspend the beads in 10 µL of TN buffer.
 

SECTION: PCR cleanup
18. Incubate at Room temperature for 2 min.

SECTION: PCR cleanup
19. Place the tube on a magnetic rack for 2 min .

SECTION: PCR cleanup
20. Transfer the eluate to a new tube.

SECTION: DNA quantification
21. Warm QuantiFluor ONE dsDNA dye to Room temperature .

SECTION: DNA quantification
22. Add 1 µL of eluted sample to 200 µL of QuantiFluor ONE dsDNA dye in 0.5 mL tube.

SECTION: DNA quantification
23. Mix thoroughly by vortexing.
SECTION: DNA quantification
24. Incubate at Room temperature for 5 min, protected from light.

SECTION: DNA quantification
25. Measure fluorescence using the Quantus Fluorometer to quantify DNA concentration.

 

SECTION: Sequencing library preparation
26. Pool all barcoded amplicons to a total of 50-100 fmoles in 10 µL of TN buffer.
 

 

SECTION: Sequencing library preparation
27. Add 1 µL of Rapid Adapter (RAP) and mix gently by pipetting.
 

SECTION: Sequencing library preparation
28. Incubate at Room temperature for 5 min.

SECTION: Sequencing library preparation
29. Store the library on ice until ready to load.

SECTION: Flow cell check
30. Open the MinION lid and insert the flow cell under the clip.

 

SECTION: Flow cell check
31. Perform flow cell check.
SECTION: Flow cell check
32. Check the number of active pores available for the experiment.
SECTION: Sample loading
33. Prepare flow cell priming mix and vortex thoroughly.
 

SECTION: Sample loading
36. Load 800 µL of the priming mix (from Step 33) into the flow cell via the priming port.
 



SECTION: Sample loading
38. Prepare the sequencing library for loading.
 

 

SECTION: Sample loading
39. Lift the SpotON sample port cover of the flow cell.
SECTION: Sample loading
41. Gently mix the sequencing library (75 µL, prepared in Step 38) by pipetting just prior to loading.

SECTION: Sample loading
42. Load the library into the flow cell via the SpotON sample port in a dropwise fashion.
 

SECTION: Nanopore sequencing
44. Start sequencing run.
 

SECTION: PCR cleanup
14. Wash the beads with 70% ethanol as follows (2/2).
SECTION: PCR cleanup
13.1. Keeping on the magnetic rack, add 200 µL of 70% ethanol without disturbing the bead pellet.
SECTION: PCR cleanup
13.2. Discard the supernatant.
SECTION: PCR cleanup
14.1. Keeping on the magnetic rack, add 200 µL of 70% ethanol without disturbing the bead pellet.
SECTION: PCR cleanup
14.2. Discard the supernatant.
SECTION: Sample loading
34. Open the priming port cover of the flow cell.
SECTION: Sample loading
35. Remove air bubbles under the cover as follows (if any).
SECTION: Sample loading
35.1. Set the volume of P1000 micropipette to 200 µL.
SECTION: Sample loading
35.2. Insert the tip into the priming port.
SECTION: Sample loading
35.3. Turn the wheel of the pipette slowly to increase the volume and draw back 20-30 µL of the buffer.
 


SECTION: Sample loading
37. Wait for 5 min.

SECTION: Sample loading
40. Load 200 µL of the priming mix (from Step 33) into the flow cell via the priming port (caution: not the SpotON sample port).
 


SECTION: Sample loading
43. Replace the SpotON sample port cover and close the priming port.
SECTION: Flushing a flow cell
45. Stop sequencing run.
SECTION: Flushing a flow cell
46. Prepare flow cell wash mix and gently mix by pipetting.
 

SECTION: Flushing a flow cell
47. Remove fluid in the waste channel via the waste port.
 

SECTION: Flushing a flow cell
48. Open the priming port cover of the flow cell.
SECTION: Flushing a flow cell
49. [Optional] If necessary, remove air bubbles under the cover by following the procedure in Step 35.
SECTION: Flushing a flow cell
50. Load 400 µL of the wash mix (from Step 46) into the flow cell via the priming port.
 

SECTION: Flushing a flow cell
51. Close the priming port.
SECTION: Flushing a flow cell
52. Wait for 60 min  to digest remaining DNA on the flow cell.

SECTION: Flushing a flow cell
53. Remove fluid in the waste channel via the waste port.
 

SECTION: Flushing a flow cell
54. Open the priming port cover of the flow cell.
SECTION: Flushing a flow cell
55. [Optional] If necessary, remove air bubbles under the cover by following the procedure in Step 35.
SECTION: Flushing a flow cell
56. Load 500 µL of Storage Buffer (S) into the flow cell via the priming port.
 


SECTION: Flushing a flow cell
57. Close the priming port.
SECTION: Flushing a flow cell
59. Store the flow cell at 4 °C for subsequent use.
SECTION: Flushing a flow cell
58. Remove fluid in the waste channel via the waste port.
 

SECTION: PCR cleanup
20.1. [Optional] Analyze 1 µL of the purified sample by gel electrophoresis to confirm the recovery.