TITLE: Sequencing fungal metabarcode with PCR primer based barcoding and Nanopore
AUTHORS: Abigail Graetz, Ashley Jones, Benjamin Schwessinger

[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 PCR barcoding of the Internal Transcribed Spacer, ITS using primers that contain barcodes, followed by ligation based amplicon sequencing with Nanopore. The initial steps of repeated PCR with high-fidelity polymerase and purification of the PCR amplicons has been performed by your demonstrator due to time limitations. We selected DNA samples of three research groups in week 5 so that we have three replicates per treatment group. This gives us 15 PCR reactions plus 1 negative control. We skip the extraction control for this practical mini-research project. This is step 1 of the protocol.

One group will perform the library preparation on the PCR barcoded and equimolar pooled amplicons. Most of the class need not be present for this library preparation [Step 2] but will join in for loading the final library and starting sequencing runs. We will also explain more about the theory during the lab. 

This protocol is applicable for week 6. 


Conceptual overview:


 

Repeat PCR reactions on the samples of three research groups using primers containing barcodes in them (see below).
Clean and pool amplicons at equimolar ratios.
End-prep the pooled amplicons to make them ready to ligate sequencing adapters.
Ligate sequencing adaptars with DNA ligase.
Make amplicon library ready for loading.
Prepare for loading onto the flowcell.
Prime flowcell to make it ready for loading.
Load library.
Start of sequencing run.
Basecall during or after the sequencing run.


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 :).

Primers used:

We used ITS primers that directly include DNA barcodes as follows, where N15-16 represents a unique barcode sequence

ITSFor (a.k.a. ITS1-F_KYO2_BARCODE): 5'-N15-16-TAGAGGAASTAAAAGTCGTAA-3'
ITSRev (a.k.a.LR6_BARCODE): 5'-N15-16-CGCCAGTTCTGCTTACC-3'

References:
Ohta, Nishi, Hirota and Matsuo, 2023, 'DNA metabarcoding workflow utilising nanopore long-read sequencing and consensus generation for rapid identification of fungal taxa with high phylogenetic resolution, BioRxiv, doi: https://doi.org/10.1101/2023.04.14.536971 
Hebert, P.D.N., Floyd, R., Jafarpour, S., Prosser, S.W.J., 2023. Barcode 100K Specimens: In a
Single Nanopore Run, BioRxiv, doi: https://doi.org/10.1101/2023.11.29.569282

[GUIDELINES]
This protocol describes how to carry out native barcoding of PCR amplicons that already contain barcodes using the Ligation Sequencing Kit (SQK-RBKLSK114). The PCR barcoding allows for 10-100,000 barcoded samples to be sequenced in one reaction. We will barcode 16 PCR amplicons

This approach is recommended for users who:
wish to multiplex 10 to 10,000 samples to reduce sequencing cost per sample
want to avoid costly (time and money) native barcoding reactions and two step PCR barcoding reactions
want to conserve amplicon length 

As always you need to bring a lab notebook, a printed version of this protocol, and a pen to record your adventures in the lab.

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 of input DNA
1. This first step has been performed by a demonstrator using DNA from the groups we selected in week 5. We selected three research groups so that we have three replicates per treatment group. This gives us 15 PCR reactions plus 1 negative control. We skip the extraction control for this practical mini-research project.

We repeated the PCR like here PCR reaction of marker regions (a.k.a metabarcodes) (protocols.io) for these samples with the two following adjustments:

We used proof-reading high fidelity polymerase like Q5 for the PCR reaction.
We used ITS primers that directly include DNA barcodes as follows, where N15-16 represents a unique barcode sequence

ITSFor (a.k.a. ITS1-F_KYO2_BARCODE): 5'-N15-16-TAGAGGAASTAAAAGTCGTAA-3'
ITSRev (a.k.a.LR6_BARCODE): 5'-N15-16-CGCCAGTTCTGCTTACC-3'

The PCR products were checked by agarose gel electrophoresis. These confirmed PCR products were then treated as follows.
SECTION: Preparation of input DNA
1.1. Purify your PCR amplicons with magnetic beads (e.g. AMPure) by adding an equal volume of magnetic beads to the PCR reaction. 

For example if your PCR reaction was 50 ul add 50 ul of beads.
SECTION: Preparation of input DNA
1.2. Incubate while rotating the tube for 10 minutes at room temperature
SECTION: Preparation of input DNA
1.3. Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.
SECTION: Preparation of input DNA
1.4. Keep the tube on the magnet and wash the beads with 200 ul of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
SECTION: Native amplicon library preparation
2. These steps will be performed by a single group we selected in week 5. 
SECTION: Native amplicon library preparation
2.2. In a 0.2 ml thin-walled PCR tube, mix the following:


 

SECTION: Native amplicon library preparation
2.3. Thoroughly mix the reaction by gently pipetting and briefly spinning down.

SECTION: Native amplicon library preparation
2.1. Program the thermal cycler:  20°C for 5 minutes and 65°C for 5 minutes. Ramp it up and keep it at 20°C so we can start the program immediately. 
SECTION: Native amplicon library preparation
2.4. Incubate the tubes at 20°C for 5 minutes and 65°C for 5 minutes using the PCR machine we already set up. Once done, briefly put the tubes  on ice to cool.
SECTION: Native amplicon library preparation
2.5. Resuspend the magentic beads for DNA binding by vortexing.
SECTION: Native amplicon library preparation
2.6. Add an equal volume of 60 ul of magnetic beads to End-prep reaction.
SECTION: Native amplicon library preparation
2.7. Incubate while rotating the tube with your hand for 5 minutes at room temperature
SECTION: Native amplicon library preparation
2.8. Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.

A demonstrator will help you with this step.
SECTION: Native amplicon library preparation
2.9. Keep the tube on the magnet and wash the beads with 200ul of freshly prepared 80% ethanol without disturbing the pellet. Remove the ethanol using a pipette and discard.
SECTION: Native amplicon library preparation
2.10. Repeat the previous step 2.9.
SECTION: Native amplicon library preparation
2.11. Briefly spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for 1 - 2 mins, but do not dry the pellet to the point of cracking
SECTION: Native amplicon library preparation
2.12. Remove the tube from the magnetic rack and resuspend the pellet in 61 μl nuclease free water.
SECTION: Native amplicon library preparation
2.13. Incubate for 3 minutes while warming the tube with your fingers.
SECTION: Native amplicon library preparation
2.14. Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
SECTION: Native amplicon library preparation
2.15. Remove and retain the full volume of eluate into a clean 1.5ml Eppendorf PCR tube.
SECTION: Native amplicon library preparation
2.16. Now we will add the sequence adapters to the End-prepped PCR amplicons


 

SECTION: Native amplicon library preparation
2.17. Thoroughly mix the reaction by gently pipetting and briefly spinning down.

SECTION: Native amplicon library preparation
2.19. Add 55 µl of resuspended magnetic beads to the reaction and mix by flicking the tube.
SECTION: Native amplicon library preparation
2.20. Incubate while rotating the tube with your hand for 5 minutes at room temperature
SECTION: Native amplicon library preparation
2.21. Spin down the sample and pellet on a magnet. Keep the tube on the magnet, and pipette off the supernatant.

A demonstrator will help you with this step.
SECTION: Native amplicon library preparation
2.22. Wash the beads by adding 250 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: Flowcell preparation for sequencing aka priming
4. These steps will be performed by one group member while the rest of the class is watching.

If you are not familiar with MinION flowcells check out this explanation video here Community - Flow cell introduction (nanoporetech.com). 

This is a useful video for flowcell priming and loading Community - Priming and loading your flow cell (nanoporetech.com).
SECTION: Flowcell preparation for sequencing aka priming
4.1. Perform a flowcell check as shown here Community - MinKNOW: Flow cell check (nanoporetech.com)
SECTION: Flowcell preparation for sequencing aka priming
4.2. To prepare the flow cell priming mix with BSA, combine the following reagents in a fresh 1.5 ml Eppendorf DNA LoBind tube. Mix by inverting the tube and pipette mix at room temperature.



 



SECTION: Flowcell preparation for sequencing aka priming
4.3. 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. This step might have been already performed if you did the flow cell check.


 

SECTION: Flowcell preparation for sequencing aka priming
4.4. Slide the flow cell priming port cover clockwise to open the priming port. The priming port cover has the Nanopore logo.


 

 

SECTION: Flowcell preparation for sequencing aka priming
4.5. IMPORTANT
Take care when drawing back buffer from the flow cell. Do not remove more than 20-30 μl, and make sure that the array of pores are covered by buffer at all times. Introducing air bubbles into the array can irreversibly damage pores.
SECTION: Flowcell preparation for sequencing aka priming
4.6. After opening the priming port, check for a small air bubble under the cover. Draw back a small volume to remove any bubbles:
Set a P1000 pipette to 200 μl
Insert the tip into the priming port
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.

Note: Visually check that there is continuous buffer from the priming port across the sensor array.


 

SECTION: Flowcell preparation for sequencing aka priming
4.7. Load 800 μl of the priming mix into the flow cell via the priming port, avoiding the introduction of air bubbles. This is best done by turning the wheel slowly and ejecting the priming buffer slowly into the priming port. Close the priming port cover during incubation. Wait for five minutes. 
 

SECTION: Flowcell preparation for sequencing aka priming
4.8. The flowcell is now ready for loading and sequencing.
SECTION: Loading of DNA library onto MinION flowcell
5. Now we will load the DNA library from step 3.23 to start sequencing. 

This is a useful video for flowcell priming and loading Community - Priming and loading your flow cell (nanoporetech.com).
SECTION: Loading of DNA library onto MinION flowcell
5.1. IMPORTANT
The Library Beads (LIB) tube contains a suspension of beads. These beads settle very quickly. It is vital that they are mixed immediately before use.
SECTION: Loading of DNA library onto MinION flowcell
5.2. Complete the flow cell priming:
priming port cover.
Gently lift the SpotON sample port cover to make the SpotON sample port accessible.
Load 200 μl of the priming mix into the flow cell priming port (not the SpotON sample port), avoiding the introduction of air bubbles.


 

SECTION: Loading of DNA library onto MinION flowcell
5.3. Mix the prepared library gently by pipetting up and down just prior to loading.
SECTION: Loading of DNA library onto MinION flowcell
5.4. Add 75 μl of the prepared 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.

A demonstrator will assist you with this step.


 

SECTION: Loading of DNA library onto MinION flowcell
5.5. Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port and close the priming port.


 

SECTION: Loading of DNA library onto MinION flowcell
5.6. IMPORTANT
Install the light shield on your flow cell as soon as library has been loaded for optimal sequencing output.
We recommend leaving the light shield on the flow cell when library is loaded, including during any washing and reloading steps.

The shield can be removed when the library has been removed from the flow cell. Gently replace the SpotON sample port cover, making sure the bung enters the SpotON port and close the priming port.

SECTION: Loading of DNA library onto MinION flowcell
5.7. Place the light shield onto the flow cell, as follows:
Carefully place the leading edge of the light shield against the clip.
Note: Do not force the light shield underneath the clip.
Gently lower the light shield onto the flow cell. The light shield should sit around the SpotON cover, covering the entire top section of the flow cell.


 

SECTION: Loading of DNA library onto MinION flowcell
5.8. Now we are ready for DNA sequencing via the MinKNOW graphical user interface.
SECTION: DNA sequencing and basecalling
6. The demonstrators will help setting up the sequencing runs. We will perform basecalling later on.

Here is a list of useful videos:
Introduction to the MinKNOW graphic user interface Community - MinKNOW: The graphical user interface (GUI) (nanoporetech.com)
How to set up a sequencing experiment Community - MinKNOW: Setting up a new experiment (nanoporetech.com)
How to assess your run is going fine Community - MinKNOW: Assessing your run (nanoporetech.com)
How MinKNOW selects the best channels Community - MinKNOW: Mux scan and active channel selection (nanoporetech.com)
More about checking on if your sequencing run is going smoothly Community - MinKNOW: Pore occupancy (nanoporetech.com)
SECTION: DNA sequencing and basecalling
6.1. We will share sequencing results and initial analysis beginning of week 7. 
SECTION: Preparation of input DNA
1.5. Repeat the previous step 1.4.
SECTION: Preparation of input DNA
1.9. Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.
SECTION: Preparation of input DNA
1.8. Incubate for 10 minutes.
SECTION: Preparation of input DNA
1.7. Remove the tube from the magnetic rack and resuspend the pellet in 20 ul of nuclease free water.
SECTION: Preparation of input DNA
1.6. Briefly spin down and place the tube back on the magnet. Pipette off any residual ethanol. Allow to dry for 1 - 2 mins, but do not dry the pellet to the point of cracking.
SECTION: Preparation of input DNA
1.12. Now we need to combine the 16 purified PCR amplicons [3 reps * 5 treatment groups + 1 negative PCR control] at equal molar ratios. We need about 400 ng DNA in total as input for the library prep as this equates to ~ 0.2 pmol when assuming an average long-ITS PCR amplicon length of 3000 bp.

This implies that we need to combine 25 ng per purified PCR amplicon and make it up to 50 ul total. These 50 ul contain our PCR amplicons in equal molar ratios and are the staring point for our library preparations.

To note is that we expect very low amounts for the negative PCR control and that it is best add as much as possible up to 25 ng to the DNA amplicon pool.
SECTION: Preparation of input DNA
1.11. Quantify 1ul of purified amplicon using Qubit BR as described in previous protocols. Note down the concentration.
SECTION: Preparation of input DNA
1.10. Remove and retain the full volume of eluate into a clean 200ul PCR tube.
SECTION: Native amplicon library preparation
2.24. Remove the tube from the magnetic rack and resuspend the pellet in 15 µl Elution Buffer (EB). Spin down and incubate for 5 minutes at room temperature.
SECTION: Native amplicon library preparation
2.23. Repeat the previous step 2.22.
SECTION: Native amplicon library preparation
2.18. Incubate the reaction for 10 minutes at room temperature.
SECTION: Native amplicon library preparation
2.27. Quantify 1ul of purified amplicon using Qubit BR as described in previous protocols. Note down the concentration and calculate the volume you need to obtain 20 fmol assuming an average amplicon length of ~ 3000 bp.

Remember:

n = c*V with n being the amount in ng, c the concentration in ng/ul, and V the volume in ul.

and use the following online calculator.

Biomath Calculators | DNA Calculator | Vector Insert Ratio (promega.com.au)
SECTION: Native amplicon library preparation
2.25. Pellet the beads on a magnet until the eluate is clear and colourless, for at least 1 minute.

SECTION: Native amplicon library preparation
2.26. Remove and retain 15 µl of eluate containing the DNA library into a clean 1.5 ml Eppendorf DNA LoBind tube.
SECTION: Native amplicon library preparation
3. We now have our library ready to load and will wait for the rest of the class to come in.
SECTION: Native amplicon library preparation
2.29. Add 37.5 uL of Sequencing Buffer (SB) and 25.5uL of Library Beads (LIB) to the sample.


Note – the library beads settle on the bottom of the tube very quickly. Make sure to mix well before pipetting. 
SECTION: Native amplicon library preparation
2.28. Make up 20 fmol of final library in 12 ul of Elution Buffer.