TITLE: Sequencing fungal and bacterial metabarcodes with native barcoding and Nanopore
AUTHORS: Carlos Goller, Benjamin Schwessinger, Carly Sjogren

[DESCRIPTION]
This protocols is part of the ANU Biosecurity mini-research project #1 "Plant Pathogen Diagnostics: Visuals, subcultures, and genomics". 

You will be provided four pots of 3-4 week old wheat plants that have been infected with different wheat pathogens. Each pot has been infected with one major pathogen. You will not know which pot has been infected with which pathogen. However, you will be provided a compendium of 10-15 wheat pathogens that will guide you to identify the infective agent for each treatment group. The fifth treatment group will be uninfected wheat plants which will be clearly identified. You can use treatment group #5 as negative control for your experiments.

In total, each group will obtain five pots each:
 
 
This specific protocol describes the molecular biology and a step-by-step guide for native barcoding and Nanopore sequencing of the metabarcode regions for fungi (Internal Transcribed Spacer, ITS) and bacteria (16S locus). Most of the steps will have been conducted by your demonstrators due to time limitations. During class we will have the final library ready to load. 

This protocol is applicable for week 5. 

Conceptual overview:

Clean up of PCR products with magnetic beads to remove buffer, enzyme, and primers.
Measure of PCR product concentration after clean up.
Start of library preparation; end-prep to repair the ends of each amplicon for ligation of barcodes including clean up with magnetic beads.
Barcode ligation. Each PCR amplicon from each group will receive a unique DNA barcode. This barcode can be used to demultiplex each sample after the sequencing run while being able to run up to 96 samples within the same reaction.
Clean up with magnetic beads to remove buffer, enzyme, and non-ligated barcodes.
Pooling of all barcoded amplicons.
Ligation of sequencing adapters.
Clean up with magnetic beads to remove buffer, enzyme, and non-ligated sequencing adapters.
Final library preparation for loading.
Flow-cell priming to be ready to load.
Library loading.
Start of sequencing run.

Demonstrators will perform step 1-8. In class we will perform step 9-12 for two libraries. One library is multiplexed to contain all ITS amplicons from class, and the other library contains all 16S amplicons from class.

You can cite this protocol in the methods section of your report as for all other protocols. No need to write it all up again :).
 

[BEFORE_START]
You must study the protocol carefully before you start. If anything is unclear post questions directly here on protocols.io.

This protocol is mostly for reference purposes and we will demonstrate all steps during practical session in class.

[GUIDELINES]
This protocol describes how to carry out native barcoding of amplicons using the Native Barcoding Kit 96 (SQK-NBD112.96). There are 96 unique barcodes available, allowing the user to pool up to 96 different samples in one sequencing experiment. 

Native Barcoding Kit  features
This kit is recommended for users who:
wish to multiplex up to 96 samples to reduce sequencing cost per sample
need a PCR-free method of multiplexing
require control over read length
would like to utilise upstream processes such as size selection or whole genome amplification

You must have read, understood, and follow the health and safety instructions provided in the "Overview Mini-Research Project #1 BIOL3106/6106" provided on Wattle (ANU learning portal). 

You must have signed and returned one copy of the "Student Safety Declaration Form For Practical Class Work" before starting any laboratory work. 

You must have read and understood the Hazard Sheets (Risk assessment) of all chemicals listed bellow in the "Safety Warnings" section. These Hazard Sheets are provided on Wattle as part of the  "Overview Mini-Research Project #1 BIOL3106/6106" document.

[STEPS]
SECTION: Preparation Before Starting
1. Prepare for your experiment (done bye the demonstrators for you).
SECTION: Preparation Before Starting
1.1. Purify your PCR amplicons with AMPure beads or similar and measure concentrations (Step 1+2 in Abstract Overview).
SECTION: Preparation Before Starting
1.2. Ensure you have your sequencing kit, the correct equipment and third-party reagents.
SECTION: Preparation Before Starting
1.3. Download the software for acquiring and analysing your data including MiniKNOW.
SECTION: Preparation Before Starting
1.4. Check your flow cell to ensure it has enough pores for a good sequencing run.
SECTION: Preparation Before Starting
2. Prepare your library
  

SECTION: Preparation Before Starting
2.1. Repair the DNA, and prepare the DNA ends for adapter attachment (Step 3 in Abstract Overview).
SECTION: Preparation Before Starting
2.2. Ligate Native barcodes supplied in the kit to the DNA ends  (Step 4+5 in Abstract Overview)..
SECTION: Preparation Before Starting
2.3. Ligate sequencing adapters supplied in the kit to the DNA ends  (Step 6-8 in Abstract Overview)..
SECTION: Preparation Before Starting
2.4. Prime the flow cell, and load your DNA library into the flow cell  (Step 9-11 in Abstract Overview)..
SECTION: Preparation Before Starting
3. Sequencing  (Step 12 in Abstract Overview).
SECTION: Preparation Before Starting
3.1. Start a sequencing run using the MinKNOW software, which will collect raw data from the device and convert it into basecalled reads.
SECTION: DNA repair and end-prep (Step 3 in Abstract Overview)
5. Prepare the NEBNext Ultra II End repair / dA-tailing Module reagents in accordance with manufacturer’s instructions, and place on ice.

 



 

SECTION: DNA repair and end-prep (Step 3 in Abstract Overview)
6. Thaw the AMPure XP Beads (AXP) at room temperature and mix by vortexing.
SECTION: DNA repair and end-prep (Step 3 in Abstract Overview)
7. In clean 0.2 ml thin-walled PCR tubes, prepare your DNA sample:
For >4 barcodes, aliquot 25-200 ng per sample


SECTION: DNA repair and end-prep (Step 3 in Abstract Overview)
8. Make up each sample to 12 µL  using nuclease-free water. Mix gently by pipetting and spin down.

SECTION: DNA repair and end-prep (Step 3 in Abstract Overview)
9. Combine the following components per sample:
Between each addition, pipette mix 10 - 20 times.
SECTION: DNA repair and end-prep (Step 3 in Abstract Overview)
9.1. 
 

 

SECTION: DNA repair and end-prep (Step 3 in Abstract Overview)
10. Combine 12 ul of sample and 3ul of reaction master mix for 15 ul reactions. Mix well by pipetting and spin down in a centrifuge.
SECTION: DNA repair and end-prep (Step 3 in Abstract Overview)
11. Using a thermal cycler, incubate at 20 °C  for 5 min and 65 °C  for 5 min .

SECTION: DNA repair and end-prep (Step 3 in Abstract Overview)
12. Transfer the sample to a clean 1.5 ml Eppendorf DNA LoBind tube.
SECTION: DNA repair and end-prep (Step 3 in Abstract Overview)
13. Resuspend the AMPure XP Beads (AXP) by vortexing.
SECTION: DNA repair and end-prep (Step 3 in Abstract Overview)
14. Add 15 µL  of resuspended AMPure XP Beads (AXP) to each end-prep reaction and mix by flicking the tube.

SECTION: DNA repair and end-prep (Step 3 in Abstract Overview)
15. Incubate on a Hula mixer (rotator mixer) for 5 min at Room temperature .

SECTION: DNA repair and end-prep (Step 3 in Abstract Overview)
16. Prepare 500 µL of fresh 70% ethanol in nuclease-free water.

SECTION: DNA repair and end-prep (Step 3 in Abstract Overview)
17. Spin down the samples and pellet the beads on a magnet until the eluate is clear and colourless. Keep the tubes on the magnet and pipette off the supernatant.
SECTION: DNA repair and end-prep (Step 3 in Abstract Overview)
18. Keep the tube on the magnet and wash the beads with 200 µL of freshly prepared 70% ethanol without disturbing the pellet. Wait for the beads to migrate towards the magnet and form a pellet. Remove the ethanol using a pipette and discard.

SECTION: DNA repair and end-prep (Step 3 in Abstract Overview)
19. Repeat the previous step.
SECTION: DNA repair and end-prep (Step 3 in Abstract Overview)
20. Briefly spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for 30 s .

 


SECTION: DNA repair and end-prep (Step 3 in Abstract Overview)
21. Remove the tube from the magnetic rack and resuspend the pellet in 10 µL  nuclease-free water. Spin down and incubate for 2 min at Room temperature .

SECTION: DNA repair and end-prep (Step 3 in Abstract Overview)
22. Pellet the beads on a magnet until the eluate is clear and colourless.
SECTION: DNA repair and end-prep (Step 3 in Abstract Overview)
23. Remove and retain 10 µL  of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

 

SECTION: DNA repair and end-prep (Step 3 in Abstract Overview)
24. Take forward equimolar mass of samples to be barcoded and pooled forward into the native barcode step. However, at this point it is also possible to store the sample at 4 °C   overnight.

SECTION: Native Barcode Ligation (Step 4-6 in Abstract Overview)
26. Prepare third party reagents in accordance with manufacturer's instructions, and place on ice.
SECTION: Native Barcode Ligation (Step 4-6 in Abstract Overview)
27. Thaw the native barcodes at Room temperature . Use one barcode per sample. Individually mix the barcodes by pipetting, spin down, and place them on ice.

SECTION: Native Barcode Ligation (Step 4-6 in Abstract Overview)
28. Select a unique barcode for every sample to be run together on the same flow cell. Up to 96 samples can be barcoded and combined in one experiment.
SECTION: Native Barcode Ligation (Step 4-6 in Abstract Overview)
29. In clean 0.2 ml thin-walled PCR tubes, add the reagents in the following order per sample: Between each addition, pipette mix 10 - 20 times.

 

SECTION: Native Barcode Ligation (Step 4-6 in Abstract Overview)
30. Ensure the components are thoroughly mixed by pipetting and spin down briefly.
SECTION: Native Barcode Ligation (Step 4-6 in Abstract Overview)
31. Incubate for 20 min at Room temperature .

SECTION: Native Barcode Ligation (Step 4-6 in Abstract Overview)
32. Add 2 µL of EDTA to each tube and mix thoroughly by pipetting and spin down briefly.

 


SECTION: Native Barcode Ligation (Step 4-6 in Abstract Overview)
33. Pool the barcoded samples in a clean 1.5 ml Eppendorf DNA LoBind tube.
 



SECTION: Native Barcode Ligation (Step 4-6 in Abstract Overview)
34. Resuspend the AMPure XP Beads (AXP) by vortexing.
SECTION: Native Barcode Ligation (Step 4-6 in Abstract Overview)
35. Add AMPure XP Beads (AXP) to the pooled reaction, and mix by pipetting for a 0.7X volume clean up.
SECTION: Native Barcode Ligation (Step 4-6 in Abstract Overview)
36. Incubate on a Hula mixer (rotator mixer) for 10 min at Room temperature .

SECTION: Native Barcode Ligation (Step 4-6 in Abstract Overview)
37. Prepare 2 mL of fresh 70% ethanol in nuclease-free water.

SECTION: Native Barcode Ligation (Step 4-6 in Abstract Overview)
38. Spin down the sample and pellet on a magnet for 5 min  . Keep the tube on the magnetic rack until the eluate is clear and colourless, and pipette off the supernatant.

SECTION: Native Barcode Ligation (Step 4-6 in Abstract Overview)
39. Keep the tube on the magnetic rack and wash the beads with 700 µL of freshly prepared 70% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.

SECTION: Native Barcode Ligation (Step 4-6 in Abstract Overview)
40. Repeat the previous step.
SECTION: Native Barcode Ligation (Step 4-6 in Abstract Overview)
41. Spin down and place the tube back on the magnetic rack. Pipette off any residual ethanol. Allow to dry for ~30 s .
 


SECTION: Native Barcode Ligation (Step 4-6 in Abstract Overview)
42. Remove the tube from the magnetic rack and resuspend the pellet in 35 µL nuclease-free water. Incubate for 10 mins.

SECTION: Native Barcode Ligation (Step 4-6 in Abstract Overview)
43. Pellet the beads on a magnetic rack until the eluate is clear and colourless.
SECTION: Native Barcode Ligation (Step 4-6 in Abstract Overview)
44. Remove and retain 35 µL of eluate into a clean 1.5 ml Eppendorf DNA LoBind tube.

SECTION: Adapter ligation and clean-up (Step 7-8 in Abstract Overview)
47. Thaw the Elution Buffer (EB) and NEBNext Quick Ligation Reaction Buffer (5x) at Room temperature , mix by vortexing, spin down and place on ice. Check the contents of each tube are clear of any precipitate.

SECTION: Adapter ligation and clean-up (Step 7-8 in Abstract Overview)
48. Spin down the Quick T4 Ligase and the Adapter Mix II H (AMII H), and place on ice.
SECTION: Adapter ligation and clean-up (Step 7-8 in Abstract Overview)
49. To deplete un-ligated sequencing adapters, thaw one tube of Short Fragment Buffer (SFB) at Room temperature , mix by vortexing, spin down and place on ice.

SECTION: Adapter ligation and clean-up (Step 7-8 in Abstract Overview)
50. In a 1.5 ml Eppendorf LoBind tube, mix in the following order:

 

 

SECTION: Adapter ligation and clean-up (Step 7-8 in Abstract Overview)
51. Ensure the components are thoroughly mixed by pipetting and spin down briefly.
SECTION: Adapter ligation and clean-up (Step 7-8 in Abstract Overview)
52. Incubate the reaction for 20 min at Room temperature .
 


SECTION: Adapter ligation and clean-up (Step 7-8 in Abstract Overview)
53. Resuspend the AMPure XP Beads (AXP) by vortexing.
SECTION: Adapter ligation and clean-up (Step 7-8 in Abstract Overview)
54. Add 20 µL of resuspended AMPure XP Beads (AXP) to the reaction and mix by pipetting.

SECTION: Adapter ligation and clean-up (Step 7-8 in Abstract Overview)
55. Incubate on a Hula mixer (rotator mixer) for 10 min  at Room temperature 

SECTION: Adapter ligation and clean-up (Step 7-8 in Abstract Overview)
56. Spin down the sample and pellet on the magnetic rack. Keep the tube on the magnet and pipette off the supernatant.
SECTION: Adapter ligation and clean-up (Step 7-8 in Abstract Overview)
57. Wash the beads by adding either 125 µL Short Fragment Buffer (SFB). Flick the beads to resuspend, spin down, then return the tube to the magnetic rack and allow the beads to pellet. Remove the supernatant using a pipette and discard

SECTION: Adapter ligation and clean-up (Step 7-8 in Abstract Overview)
58. Repeat the previous step.
SECTION: Adapter ligation and clean-up (Step 7-8 in Abstract Overview)
59. Spin down and place the tube back on the magnet. Pipette off any residual supernatant. Allow to dry for ~30 s 
 


SECTION: Adapter ligation and clean-up (Step 7-8 in Abstract Overview)
60. Remove the tube from the magnetic rack and resuspend the pellet in 15 µL of Elution Buffer (EB).

SECTION: Adapter ligation and clean-up (Step 7-8 in Abstract Overview)
61. Spin down and incubate for 10 min at 37 °C  . Periodically agitate the sample by gently flicking to encourage DNA elution

SECTION: Adapter ligation and clean-up (Step 7-8 in Abstract Overview)
62. Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 min 

SECTION: Adapter ligation and clean-up (Step 7-8 in Abstract Overview)
63. Remove and retain 15 µL of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube.

 

 


SECTION: Priming and loading the SpotON flow cell (Step 9-11 in Abstract Overview)
65. We recommend all new users watch the 'Priming and loading your flow cell' video before your first run.

We recommend using the Loading Beads II (LBII) for loading your library onto the flow cell for most sequencing experiments. However, if you have previously used water to load your library, you must use Loading Solution (LS) instead of water
SECTION: Priming and loading the SpotON flow cell (Step 9-11 in Abstract Overview)
66. Thaw the Sequencing Buffer II (SBII), Loading Beads II (LBII) or Loading Solution (LS, if using), Flush Tether (FLT) and one tube of Flush Buffer (FB) at Room temperature before mixing the reagents by vortexing and spin down at Room temperature 

SECTION: Priming and loading the SpotON flow cell (Step 9-11 in Abstract Overview)
67. To prepare the flow cell priming mix, add 30 µL  of thawed and mixed Flush Tether (FLT) directly to the tube of thawed and mixed Flush Buffer (FB), and mix by vortexing at Room temperature .

SECTION: Priming and loading the SpotON flow cell (Step 9-11 in Abstract Overview)
68. Open the MinION lid and slide the flow cell under the clip. Press down firmly on the flow cell to ensure correct thermal and electrical contact
  

SECTION: Priming and loading the SpotON flow cell (Step 9-11 in Abstract Overview)
70. Slide the priming port cover clockwise to open the priming port.
 

SECTION: Priming and loading the SpotON flow cell (Step 9-11 in Abstract Overview)
71. After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles (a few µl):

  

SECTION: Priming and loading the SpotON flow cell (Step 9-11 in Abstract Overview)
71.1. Set a P1000 pipette to 200 µl
SECTION: Priming and loading the SpotON flow cell (Step 9-11 in Abstract Overview)
71.2. Insert the tip into the priming port
SECTION: Priming and loading the SpotON flow cell (Step 9-11 in Abstract Overview)
71.3. Turn the wheel until the dial shows 220-230 ul, to draw back 20-30 ul, or until you can see a small volume of buffer entering the pipette tip
 

SECTION: Priming and loading the SpotON flow cell (Step 9-11 in Abstract Overview)
72. Load 800 µL  of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. Wait for 5 min . During this time, prepare the library for loading by following the steps below.
  

SECTION: Priming and loading the SpotON flow cell (Step 9-11 in Abstract Overview)
73. Thoroughly mix the contents of the Loading Beads II (LBII) by pipetting.

 

SECTION: Priming and loading the SpotON flow cell (Step 9-11 in Abstract Overview)
74. In a new tube, prepare the library for loading as follows:

 

 

SECTION: Priming and loading the SpotON flow cell (Step 9-11 in Abstract Overview)
75. Complete the flow cell priming:

SECTION: Priming and loading the SpotON flow cell (Step 9-11 in Abstract Overview)
75.1. Gently lift the SpotON sample port cover to make the SpotON sample port accessible.
SECTION: Priming and loading the SpotON flow cell (Step 9-11 in Abstract Overview)
75.2.  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: Priming and loading the SpotON flow cell (Step 9-11 in Abstract Overview)
76. Mix the prepared library gently by pipetting up and down just prior to loading.
SECTION: Priming and loading the SpotON flow cell (Step 9-11 in Abstract Overview)
77. Add 75 µL  of sample 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: Priming and loading the SpotON flow cell (Step 9-11 in Abstract Overview)
78. Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port, close the priming port and replace the MinION lid. 
  

SECTION: Ending the Experiment
80. 
 

SECTION: Ending the Experiment
81. Follow the returns procedure by washing out the flow cell ready to send back to Oxford Nanopore.
Instructions for returning flow cells can be foundhere.

 

SECTION: DNA repair and end-prep (Step 3 in Abstract Overview)
4. Materials 
25-200 ng amplicon DNA per sample for >12 barcodes.
AMPure XP Beads (AXP) 

Consumables 

NEBNext Ultra II End repair / dA-tailing Module (E7546) 
Nuclease-free water (e.g. ThermoFisher, cat # AM9937) 
Freshly prepared 70% ethanol in nuclease-free water
0.2 ml thin-walled PCR tubes 
1.5 ml Eppendorf DNA LoBind tubes 
Qubit™ Assay Tubes (ThermoFisher Q32856) 
Qubit dsDNA HS Assay Kit (ThermoFisher Q32851)

Equipment 
P1000 pipette and tips 
P100 pipette and tips
P10 pipette and tips 
Thermal cycler at 20°C and 65°C 
Ice bucket with ice 
Microfuge Hula mixer (gentle rotator mixer) 
Magnetic rack
SECTION: Native Barcode Ligation (Step 4-6 in Abstract Overview)
25. Materials 
AMPure XP Beads (AXP) 
Native Barcodes (NB01-96) 
EDTA (EDTA)

Consumables 
Nuclease-free water (e.g. ThermoFisher, cat # AM9937) 
Freshly prepared 70% ethanol in nuclease-free water 
NEB Blunt/TA Ligase Master Mix (M0367) 
1.5 ml Eppendorf DNA LoBind tubes 
2 ml Eppendorf DNA LoBind tubes 
0.2 ml thin-walled PCR tubes Qubit™ Assay Tubes (ThermoFisher Q32856) 
Qubit dsDNA HS Assay Kit (ThermoFisher Q32851)

Equipment 
Magnetic rack 
Vortex mixer 
Hula mixer (gentle rotator mixer) 
Microfuge 
Thermal cycler 
Ice bucket with ice 
P1000 pipette and tips 
P100 pipette and tips 
P10 pipette and tips

Optional Equipment 
Qubit fluorometer (or equivalent for QC check)
SECTION: Native Barcode Ligation (Step 4-6 in Abstract Overview)
45. Take forward the barcoded DNA library to the adapter ligation and clean-up step. However, at this point it is also possible to store the sample at 4 °C   .

SECTION: Adapter ligation and clean-up (Step 7-8 in Abstract Overview)
46. Materials 
Long Fragment Buffer (LFB) 
Short Fragment Buffer (SFB) Elution Buffer from the Oxford Nanopore kit (EB) 
Adapter Mix II H (AMII H) 
AMPure XP Beads (AXP)

Consumables 
Quick T4 DNA Ligase in NEBNext® Quick Ligation Module (NEB E6056) 
NEBNext® Quick Ligation Reaction Buffer (NEB B6058)
1.5 ml Eppendorf DNA LoBind tubes 
Qubit™ Assay Tubes (ThermoFisher Q32856) 
Qubit dsDNA HS Assay Kit (ThermoFisher Q32851)

Equipment 
Microfuge 
Magnetic rack
Vortex mixer 
Hula mixer (gentle rotator mixer) 
Thermal cycler 
P1000 pipette and tips 
P200 pipette and tips 
P100 pipette and tips
P20 pipette and tips 
P10 pipette and tips 
Ice bucket with ice
SECTION: Priming and loading the SpotON flow cell (Step 9-11 in Abstract Overview)
64. Materials 
Flush Buffer (FB)
Flush Tether (FLT)
Loading Beads II (LBII)
Sequencing Buffer II (SBII)
Loading Solution (LS)

Consumables
1.5 ml Eppendorf DNA LoBind tubes
Nuclease-free water (e.g. ThermoFisher, cat # AM9937)

Equipment
MinION
SpotON Flow Cell
P1000 pipette and tips
P100 pipette and tips
P20 pipette and tips
P10 pipette and tips
SECTION: Priming and loading the SpotON flow cell (Step 9-11 in Abstract Overview)
69. Complete a flow cell check to assess the number of pores available before loading the library.
This step can be omitted if the flow cell has been checked previously.
SECTION: Data Acquisition and Basecalling (Step 12 in Abstract Overview)
79. For information on starting a sequencing run in MinKNOW (with a slight different library prep) check out this video https://www.youtube.com/watch?v=oCOixfewXo4