TITLE: ForBio Course ONT Barcoding Protocol
AUTHORS: Emily Hartop

[DESCRIPTION]
Protocols for ONT Barcoding Course at NTNU August 2024

[STEPS]
SECTION: HotSHOT Extraction
3. Pipette 10-20 μL of HOTSHOT alkaline lysis reagent into wells of a microplate 
SECTION: HotSHOT Extraction
4. Remove specimens onto a clean paper towel, dab on towel before loading to remove excess ethanol
SECTION: HotSHOT Extraction
5. Load specimens and check to make sure all specimens are submerged. If necessary, carefully submerge any floating specimens with forceps. Leave the last well empty as your negative.
SECTION: HotSHOT Extraction
6. Run extraction in a thermocycler for 18 minutes at 65°C followed by 2 minutes at 98°C.
SECTION: HotSHOT Extraction
7. Remove the plate from the thermocycler and pipette in 10 μL (or whatever quantity of lysis you used) of HotSHOT Tris HCl buffer to each well. 
SECTION: HotSHOT Extraction
8. Specimens should be removed as soon as possible to avoid crystal formation.
SECTION: Preparing for PCR
10. Prepare and distribute PCR mix in plate or strip tube wells according to your needs:
SECTION: Preparing for PCR
9. Prepare plate or strip tube with 1 µL of R primer (at 10 μM) in each well (different R primer in every well).
SECTION: PCR
12. Run the thermocycler protocol according to your chosen barcode:
SECTION: Agarose Gel for QC
13. Pick the frame and combs suitable for your samples. Tape the open ends of the frame (tape is in the drawer to the left of the gel station) and position the comb.
SECTION: Agarose Gel for QC
14. Pour 100 mL of agarose gel mixture (pre-prepared in the oven) into a flask and add 10 µl of SYBR stain. Swirl to mix.
SECTION: Agarose Gel for QC
15. Pour the mixture into the frame and allow to set for 30 minutes. Submerged the gel into the buffer, ensuring it is completely covered.
SECTION: Agarose Gel for QC
16. Fill with 2 µl of PCR product/well. If you are not using dyed mastermix, use loading dye (in fridge). Ladder is also in fridge.
SECTION: Agarose Gel for QC
17. Run at 110V for 35 minutes. 
SECTION: Agarose Gel for QC
18. Turn off the electrophoresis and remove the gel from the buffer. Place it on the light panel and turn on the light, close the door. On the computer, open GeneSnap and use the green button to take a photo.
SECTION: Making HotSHOT
1. Alkaline lysis solution:
1 gNaOH
0.074 g Na2 EDTA 
1 L H2O

Combine reagents in a 1 L flask. 
SECTION: Making HotSHOT
2. Neutralizing Buffer:
6.3 g Tris HCl (do not use Tris base!)
1 L H2O

Combine reagents in a 1 L flask. 
SECTION: Bead clean-up: washing
25. Keep the tube on the magnet and wash twice with 500 µL 80 % Ethanol. Specifically:
     a. Add 500 µL of 80% ethanol, gently, to the pellet while still in the magnetic rack, wait for 20 secs, then remove and discard the ethanol.
     b. Repeat step (a), but this time be sure to remove all the ethanol (use a small pipette tip to remove the last droplets of ethanol).

SECTION: Bead clean-up: washing
26. Leave the bead pellet to dry (make sure the tube lid is open) for 10-15 mins, or until the bead pellet looks less shiny and wet.
SECTION: Bead clean-up: elution
27. Remove the plate from the magnet. To elute the DNA, add 30 µL of molecular grade water. Mix by pipetting in and out to re-suspend the beads, then vortex and briefly spin down.
SECTION: Bead clean-up: elution
28. Close the tube and incubate for 5-10 mins at room temperature (not in the magnetic rack).
SECTION: Bead clean-up: elution
29. Place tube in the magnetic rack (lid open) and wait for the solution to become clear. Remove the DNA to a clean tube, careful not to disturb the beads.
SECTION: Bead clean-up: elution
31. Run a gel with 2 µL of (a) pooled product, (b) supernatant , (c) final cleaned product
SECTION: Quantification with Qubit
32. Label 5 0.5-mL tubes for (2) standards and (3) pool samples. You will take the average ng/µl from the three replicates as the pool concentration.
Note: label only the tops of the tubes and use Qubit approved tubes.
SECTION: Quantification with Qubit
33. You will need approximately 1000 µL of working solution for the standards and samples. Make this by combining 5 µL of Qubit‱ reagent plus 995 µL of Qubit‱ buffer.
SECTION: Quantification with Qubit
34. Prepare the tubes for standards with 190 µL of working solution in each tube and 10 µL of standard. Vortex for several seconds to mix.
SECTION: Quantification with Qubit
35. Prepare the tubes for your pool samples with 199 µL of working solution in each tube and 1 µL of the pool. Vortex for several seconds to mix.
SECTION: Quantification with Qubit
36. Allow all the tubes to sit at room temperature for 2 minutes before proceeding.
SECTION: Quantification with Qubit
37. On the Qubit, choose "DNA" on the first screen, then the appropriate assay (we use dsDNA Broad Range).
SECTION: Quantification with Qubit
38. Click "Yes" at the bottom of the next screen to read new standards, then insert Standard 1 and hit "Read". It will then prompt you for Standard 2, and your samples. When you have read the last sample it will give you the average in µg/mL (ng/µL). Depending on your Qubit, this may be the concentration of your sample or the entire solution. If Qubit 2.0, it will give you the entire solution and you will need to multiply this number by 200 to get your sample concentration (if you input 1 µL).
SECTION: Library Preparation and Loading
40. Important! The following modifications to ONT protocols are made:
1. Omit the DCS from end-prep solution
2. Use 100 ng DNA as input
3. During adapter ligation, use a 1:1 ratio of beads and product

A checklist for end-prep, adapter ligation, clean-up and loading:
  
For the full protocol with detailed photos and instructions for loading:
  

SECTION: Bead clean-up: elution
30. You now have a tube with approximately 30 µL of cleaned DNA. Reserve 2 µL of this cleaned product for the gel.
SECTION: Quantification with Qubit
39. You want to load 200 ng of DNA for a MinION, 100 ng for a Flongle. To get the amount of your cleaned DNA you will need, divide the ng you need by the concentration of your sample (ng/(ng/µL)=µL (eg. 100ng/(5ng/µL)=20µL)
SECTION: Preparing for PCR
11. Add 5 µL of DNA to each well.
SECTION: After the run
80. After basecalling, you will have a lot of compressed fastq files in folders "pass" and "fail". These can be extracted using 7-zip, and then combined into one file using the cat function (requires Ubuntu if you are on Windows):
1. cd to pass folder
2. cat *.fastq > pass.fastq
3. cd to fail folder
4. cat *.fastq > fail.fastq
5. Place both the fail and pass fastq files in the same folder, make that folder you working directory, and run cat one more time to make a single fastq
SECTION: Bead clean-up: preparation, binding/separation
20. Make fresh 80% ethanol (eg. 400 mL pure ethanol + 100 mL molecular grade H2O for 500 mL 80% ethanol).
SECTION: Bead clean-up: preparation, binding/separation
21. Bring beads to room temperature and re-suspend them by vortexing thoroughly. 
SECTION: Pooling
19. Pool 3 µL of PCR product from each well, first into a strip tube and then into a vial. Remove 500 µL of pooled product to a 1.5 mL tube in preparation for bead clean-up. The remaining product can be stored in case it is needed in the future. It will also be included in the electrophoresis gel. 
SECTION: Bead clean-up: preparation, binding/separation
22. Add an equal volume (1:1) of beads to the volume of pooled PCR product (e.g. 500 µL of beads to 500 µL of pooled PCR product). Vortex briefly, then spin down in microfuge very briefly.
SECTION: Bead clean-up: preparation, binding/separation
23. Incubate for 15 min at room temperature with the tube lid open.
SECTION: Bead clean-up: preparation, binding/separation
24. Put the tube on the magnetic rack and wait until a bead pellet is formed, and the solution is clear* (5 minutes or so) . Carefully aspirate the supernatant, reserving for the electrophoresis gel.
*Blue mastermix will still be blue!
SECTION: After the run
81. ONTBarcoder is available for download here. 
It requires a demultiplexing file (see your handout) and the combined fastq to run. 
 
 

SECTION: Adapter Ligation and Clean-up
55. Spin down LA and T4 Ligase, place on ice
SECTION: Adapter Ligation and Clean-up
66. Remove the tube from the magnetic rack and resuspend pellet in 7μl EB
SECTION: Adapter Ligation and Clean-up
65. Spin down and place the tube back on the magnet. Pipette off any residual supernatant. Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking
SECTION: Adapter Ligation and Clean-up
64. Repeat the previous step
SECTION: Adapter Ligation and Clean-up
63. Wash the beads by adding 125μl SFB
Flick the beads to resuspend and briefly spin down
Return the tube to the magnetic rack and allow the beads to pellet
Remove the 125μl supernatant using a pipette and discard
SECTION: Adapter Ligation and Clean-up
62. Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant (~100μl).
Discard supernatant
SECTION: Adapter Ligation and Clean-up
61. Incubate on Hula Mixer - 5min @ room remperature
SECTION: Adapter Ligation and Clean-up
60. Resuspend beads and add 50μl to the reaction
flick and spin
SECTION: Adapter Ligation and Clean-up
59. Incubate - 10min @ room temperature
SECTION: Adapter Ligation and Clean-up
58. Into DNA Library, add:
12.5μl LNB
5μl T4 Ligase
2.5μl LA
50μl Total
Pipette mix, spin briefly
SECTION: Adapter Ligation and Clean-up
57. Thaw EB and SFB at room temperature
Vortex, spin, place on ice
SECTION: Adapter Ligation and Clean-up
56. Thaw LNB at room temperature
Spin and mix by pipetting, then place on ice
SECTION: End Preparation
41. Thaw Ultra II End-prep Reaction Buffer & Enzyme Prep on ice
Do Not Vortex End Prep Enzyme Mix - flick, invert and spin
Vortex End-prep Reaction Buffer and spin
SECTION: End Preparation
42. Transfer amplicon DNA into 0.2ml PCR tube
Adjust to 24.5μl with nuclease-free water
Flick and spin
SECTION: End Preparation
46. Heavily vortex AXP to resuspend and add 30μl to DNA sample - mix by flicking and spin
SECTION: End Preparation
45. Transfer 30μl DNA sample to clean 1.5ml Eppendorf
SECTION: End Preparation
44. Run in a thermal cycler - 5min @ 20°C, 5min @ 65°C
SECTION: End Preparation
43. Additionally, add:
3.5μl End-Prep Reaction Buffer
1.5μl End-Prep Enzyme Mix
30μl Total
Pipette mix and spin
SECTION: End Preparation
52. Remove the tube from the magnetic rack and resuspend the pellet in 30 μl nuclease-free water. Incubate for 2 minutes at room temperature.
SECTION: End Preparation
51. Spin down and place the tube back on the magnet.
Pipette off any residual ethanol. 
Allow to dry for ~30 seconds, but do not dry the pellet to the point of cracking
SECTION: End Preparation
50. Repeat previous step
SECTION: End Preparation
49. Keep the tube on the magnet and wash the beads with 200μl of freshly prepared 80% ethanol without disturbing the pellet. Remove the 200μl ethanol using a pipette and discard.
SECTION: End Preparation
48. Spin down the sample and pellet on a magnet until supernatant is clear and colourless. Keep the tube on the magnet, and pipette off the supernatant (~60μl).
Discard supernatant
SECTION: End Preparation
47. Incubate on Hula Mixer  - 5 minutes at room temperature.
SECTION: End Preparation
54. Remove and retain 30μl of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube
Dispose of pelleted beads
It is possible to store this sample at 4°C overnight
SECTION: End Preparation
53. Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute
SECTION: Adapter Ligation and Clean-up
70. Quantify 1μl of eluted sample using a Qubit fluorometer - See previous section
SECTION: Adapter Ligation and Clean-up
69. Remove and retain 7 μl of eluate containing the DNA library into a clean 1.5ml Eppendorf
Dispose of pelleted beads
SECTION: Adapter Ligation and Clean-up
68. Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute
SECTION: Adapter Ligation and Clean-up
67. Incubate for 10 minutes at room temperature
SECTION: Adapter Ligation and Clean-up
71. Make up library to 5μl at 10fmol with EB
Use a calculator such as NEBioCalculator
Mass to Moles
Basepairs are ~700 (for 1-step indexed COI amplicons)

SECTION: Loading the Flongle
74. Thaw SB, LIB, FCT and FCF @ room temp, vortex and spin
SECTION: Loading the Flongle
75. Create flow cell Primer Mix in a 1.5ml Eppendorf by adding:
117μl FCF
3μl FCT
120μl Total
Mix by pipetting
SECTION: Loading the Flongle
72. Remove Flongle from the fridge, keeping the pack sealed until it reaches room temperature
SECTION: Loading the Flongle
73. Install Flongle Adaptor into Minion, and then install the Flongle into the Adaptor and run a Flow Cell Check in MinKNOW
SECTION: Loading the Flongle
79. Seal tab, close MinION lid, start sequencing!
SECTION: Loading the Flongle
78. Add the 30μl Sequencing Mix to the flow cell, ensuring no air bubbles
SECTION: Loading the Flongle
77. Add to 5μl DNA library to create to create sequencing mix:
15μl SB
10μl LIB - vortexed immediately before use
30μl Total
SECTION: Loading the Flongle
76. Open Tab on Flongle, and withdraw any air in the port
Add Primer Mix, ensuring no air bubbles are introduced.