TITLE: PCR reaction of marker regions (a.k.a metabarcodes) for two kingdoms
AUTHORS: Abigail Graetz, 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 is a step by step guide to perform PCR reactions on the extracted DNA samples of the five TG. The two PCR reaction target conserved marker regions (a.k.a. metabarcodes) for two kingdoms. With 16S primers targeting bacteria and ITS primers targeting fungi. 

The simplified idea of metabarcodes is that one can amplify a region that is conserved enough to generate primers that allow one to amplify this region from "all" members of the kindgom somewhat representatively. Within the amplicon though there are located hypervariable regions that are distinct between different species or genera. Matching these amplicons against a databases let's one identify the organism of this specific kingdom present in the sample. 

At least this is the simple theory, however as you will see in the lectures there are many issues and biases to consider when applying these techniques in general and specifically to pathogen diagnostics for biosecurity purposes.

The final goal is to achieve the following:
To measure and adjust DNA concentration for each sample to allow for equal amount of DNA being added to each sample.
Perform PCR reaction for the fungal kingdom metabarcode using published ITS (Internal transcribed spacer) primers.
Perform PCR reaction for the bacterial kingdom metabarcode using published 16S primers.

This protocol is applicable for week 4. 

Protocols progress overview:

Week 4 PCR reaction for 16S and ITS.
 
You will measure double stranded DNA concentration with a dye based method. Here the dye binds to double stranded DNA only and this will change the fluorescent of the dye in a way directly proportional to the DNA concentration. You will use the pre-mixed broad range dye kit https://www.thermofisher.com/order/catalog/product/Q32853.

The primers for the ITS PCR reactions are called ITSFor and ITSRev. These are based on White et. al., 1989 and have the following sequences.

ITSFor (a.k.a. NS5): 5'-AACTTAAAGGAATTGACGGAAG-3'
ITSRev (a.k.a.LR6): 5'-CGCCAGTTCTGCTTACC-3'

The expected amplicon size is around 2.5 to 3 kb (kilobase pairs).

The primers for the 16S PCR reaction are called 16SFor and 16SRev. These are based on Wuyts et. al., 2002, Baker et. al., 2003, and Youssef et. al., 2009.

16SFor (a.k.a. 27F, E8F, or E8Fa): 5'-AGAGTTTGATCCTGGCTCAG-3'
16SRev (a.k.a. 1492R): 5'-ACCTTGTTACGACTT-3'
 
Importantly, these 16S primers also partially bind and amplify 16S regions found in plant mitochondria and chloroplast. The amplification of the plant 16S region has to be therefore inhibited with sequence specific probes called PNAs (Peptide mediated Nucleic Acids) https://www.pnabio.com/products/PCR_blocker.htm. 

We will use the following PNAs for the 16S PCR reactions. These are based on Lundberg et a. 2013.

The anti-mitochondrial PNA (mPNA): 5′-GGCAAGTGTTCTTCGGA-3′
The anti-plastid PNA (pPNA): 5′-GGCTCAACCCTGGACAG-3′

References:

White, Bruns, Lee and Taylor, 1989, 'Amplification and direct sequencing of fungal ribosomal RNA Genes for phylogenetics', in PCR - Protocols and Applications - A Laboratory Manual, Academic Press 
 
Wuyts, Van de Peer, Winkelmans and De Wachter, 2002, 'The European database on small subunit ribosomal RNA', Nuc. Ac. Res., vol. 30 (1), pp. 183-185  

Baker, Smith and Cowan, 2003, 'Review and re-analysis of domain-specific 16S primers', J. Microbiol. Meth., vol. 55 (3), pp. 541-555 
 
Youssef, Sheik, Krumholz, Najar, Roe and Elshahed, 2009, 'Comparison of species richness estimates obtained using nearly complete fragments and simulated pyrosequencing-generated fragments in 16S rRNA gene-based environmental surveys', App. Env. Microbiol., vol. 75 (16)
 
Lundberg, Derek S., Scott Yourstone, Piotr Mieczkowski, Corbin D. Jones, and Jeffery L. Dangl. “Practical Innovations for High-Throughput Amplicon Sequencing.” Nature Methods 10, no. 10 (October 2013): 999–1002. 
 
 
 
 
 
 
 
 
 

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

Additional protocol specific notes:

We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (98°C). All components should be mixed prior to use.
The PNAs must be heated at 55oC for 10 mins before use (this will be done for you).

[GUIDELINES]
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: Week 4: DNA quantification and dilution
1. The first section describes the quantification of extracted DNA from week 3. Once you know the concentration for each TG you can generate a DNA stock solution at a final concentration of 10 ng/ul. This stock solution for each TG will be used for the following PCR reaction and DNA quality control measures in week 5.
SECTION: Week 4: DNA quantification and dilution
2. You will receive your DNA extracts from the previous practical session in week 3. You should receive five tubes in total containing the DNA for each TG.
SECTION: Week 4: DNA quantification and dilution
3. Measure DNA concentration with Qubit broad range (BR).
SECTION: Week 4: DNA quantification and dilution
3.1. You will receive five tubes containing 198uL BR Qubit reaction ready to use. Label the tubes with treatment group name.
SECTION: Week 4: DNA quantification and dilution
3.2. For each TG, add 2uL of DNA extracted to the corresponding BR Qubit tube. Make sure to add the correct DNA sample to the correctly labelled BR tube.
SECTION: Week 4: DNA quantification and dilution
3.3. Vortex your BR Qubit tube containing the DNA.
SECTION: Week 4: DNA quantification and dilution
3.4. Collect the liquid at the bottom of the tube with a quick "hand-centrifuge".
SECTION: Week 4: DNA quantification and dilution
3.5. Incubate for 1-5 min.
SECTION: Week 4: DNA quantification and dilution
3.6. Wipe down the outside of your tube with a Kim wipe.
SECTION: Week 4: DNA quantification and dilution
3.7. Measure your concentration using the Qubit machine. Demonstrators will facilitate the measurement. 
SECTION: Week 4: DNA quantification and dilution
3.8. Note down the concentration of each TG DNA sample in ng/ul.
SECTION: Week 4: DNA quantification and dilution
5. Now for each TG, dilute the DNA sample to a concentration of 10 ng/ul with nuclease free water. Make up a DNA total stock solution of 100ul. 

The important formula here is: 

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

You want V = 100ul and c =10 ng/ul. This means you will have 1000ng in 100 ul.

The question is how much volume of the original DNA do you need to obtain 1000ngs?

Here now n = 1000ng and c equals your measured concentration. Let's assume your measured c = 56 ng/ul for an assumed sample. Than we can resolve the equation as follows.

n = c*V -> V = n/c -> 1000ng/56ng/ul =  17.85 ul

So you need to use 17.85ul in 100ul total volume to obtain 1000ng. 

This means you need to mix 17.85ul of extracted DNA (c = 56 ng/ul) and mix it with 82.15 ul nuclease free water to obtain your final 100ul @ 10 ng/ul DNA stock solution.
SECTION: Week 4: DNA quantification and dilution
5.1. For each TG, calculate the required volume of extracted DNA and nuclease free water to obtain your final 100ul @ 10 ng/ul DNA stock solution. 
SECTION: Week 4: DNA quantification and dilution
4. Label five 1.5ml tubes with your research group name, treatment group name, date, and the DNA concentration 100 ng/ul.
SECTION: Week 4: DNA quantification and dilution
5.2. Check with a demonstrator that your calculations are correct
SECTION: Week 4: DNA quantification and dilution
5.3. For each TG, mix the the required volume of extracted DNA and nuclease free water to obtain your final 100ul @ 10 ng/ul DNA stock solution. Mix the sample by pipetting up and down.
SECTION: Week 4: DNA quantification and dilution
6. You now should have five DNA stock solution tubes, one for each TG. These are the DNA solutions you will need for the PCR reactions below and the DNA quality control measures in week 5. 
SECTION: Week 4: ITS PCR reaction
7. Now you will amplify the ITS regions in the DNA samples you extracted from wheat leaves. 
You will use the DNA stock solutions and 50 ng per reaction. 
You will use a proof-reading polymerase to reduce the errors introduced during PCR. 
You will use the following primer set for amplification.

ITSFor: 5'-AACTTAAAGGAATTGACGGAAG-3'
ITSRev: 5'-CGCCAGTTCTGCTTACC-3'

SECTION: Week 4: ITS PCR reaction
7.1. Label two PCR strip tubes of 8 with your research group name, 1 to 6, and ITS. 
Note down the order 1 to 6 in your lab notebook and what they correspond to. E.g. 1 = TG1,
6= negControl.
You will use one of these strip tubes for the PCR now.
The other will be empty today but used next time for PCR quality control purposes.
SECTION: Week 4: ITS PCR reaction
7.2. Make a master mix for the ITS PCR reaction for 6.5 samples. TG1 to TG5 and negative control.
Keep master mix on ice at all times.

Mix the following amounts in the specified order to make a master mix for 6.5 reactions. 

Cross off each reagents you added with a pen to make sure you added all reagents properly.

Once you added all the reagents (1-4), close the tube, vortex it, and spin it down to collect the liquid in the bottom of the tube.


 

SECTION: Week 4: ITS PCR reaction
7.3. Aliquot 45ul of the master mix into the PCR strip tube of 8 that you labelled appropriately. Add the master mix only to the tubes labelled previously. Close all lids.
Keep the PCR strip on ice.
SECTION: Week 4: ITS PCR reaction
7.4. For each TG, add 5ul of the DNA stock solution (10ng/ul) to the correct PCR tube. Mix by pipetting up and down. Close lid once you added the correct DNA stock solution.
In the case of the negative control, add 5ul of nuclease free water instead.
Keep reactions on ice.
SECTION: Week 4: ITS PCR reaction
7.5. Close the lids and hand over your PCR strip tubes (one full and one empty) to the allocated demonstrator.
SECTION: Week 4: ITS PCR reaction
8. The demonstrator will run the following PCR reaction with specific conditions for the ITS primers and amplicon length. 

STEP 1: 98oC for 30 sec; Initial denaturation
STEP 2: 98oC for 20 sec; Denaturation
STEP 3: 59oC for 30 sec; Primer annealing
STEP 4: 72oC for 90 sec; Extension, 20-30 seconds/kb fragment size
GO TO STEP 2 for 30 additional cycles; Amplification
STEP 5: 72oC for 2 min; Final extension
STEP 6: 20oC for 1 hour Hold
SECTION: Week 4: 16S PCR reaction
10. Now you will amplify the 16S regions in the DNA samples you extracted from wheat leaves. 
You will use the DNA stock solutions and 50 ng per reaction. 
You will use a proof-reading polymerase to reduce the errors introduced during PCR. 

You will use the following primer set for amplification:

16SFor: 5'-AGAGTTTGATCCTGGCTCAG-3'
16SRev: 5'-ACCTTGTTACGACTT-3'

You will use the following PNAs set for blocking amplifcation of plant 16S sequences:

mPNA: 5′-GGCAAGTGTTCTTCGGA-3′
pPNA: 5′-GGCTCAACCCTGGACAG-3′

SECTION: Week 4: 16S PCR reaction
11. The demonstrator will run the following PCR reaction with specific conditions for the 16S primers, PNAs, and amplicon length. 

STEP 1: 98oC for 30sec; Initial denaturation
STEP 2: 98oC for 20 sec; Denaturation
STEP 3: 78oC for 10 sec; PNA annealing
STEP 4: 58oC for 30 sec; Primer annealing
STEP 5: 72oC for 45 sec; Extension, 20-30 seconds/kb fragment size
GO TO STEP 2 for 30 additional cycles; Amplification
STEP 7: 72oC for 2 min; Final extension
STEP 8: 20oC for 1 hour Hold
SECTION: Week 4: ITS PCR reaction
9. The demonstrators will aliquot out 5uL of the reaction for quality control into the empty PCR strip tube you labelled.
SECTION: Week 4: 16S PCR reaction
10.1. Label two PCR strip tubes of 8 with your research group name, 1 to 6, and 16S. 
Note down the order 1 to 6 in your lab notebook and what they correspond to. E.g. 1 = TG1,
6= negControl.
You will use one of these strip tubes for the PCR now.
The other will be empty today but used next time for PCR quality control purposes.
SECTION: Week 4: 16S PCR reaction
10.3. Aliquot 45ul of the master mix into the PCR strip tube of 8 that you labelled appropriately. Add the master mix only to the tubes labelled previously. Close all lids.
Keep the PCR strip on ice.
SECTION: Week 4: 16S PCR reaction
10.2. Make a master mix for the 16S PCR reaction for 6.5 samples. TG1 to TG5 and negative control.
Keep master mix on ice at all times.

Mix the following amounts in the specified order to make a master mix for 6.5 reactions. 

Cross off each reagents you added with a pen to make sure you added all reagents properly.

Once you added all the reagents (1-4), close the tube, vortex it, and spin it down to collect the liquid in the bottom of the tube.

 

 
 
SECTION: Week 4: 16S PCR reaction
10.4. For each TG, add 5ul of the DNA stock solution (10ng/ul) to the correct PCR tube. Mix by pipetting up and down. Close lid once you added the correct DNA stock solution.
In the case of the negative control, add 5ul of nuclease free water instead.
Keep reactions on ice.
SECTION: Week 4: 16S PCR reaction
10.5. Close the lids and hand over your PCR strip tubes (one full and one empty) to the allocated demonstrator.
SECTION: Week 4: 16S PCR reaction
12. The demonstrators will aliquot out 5uL of the reaction for quality control into the empty PCR strip tube you labelled.
SECTION: Done by demonstrator: Oxford Nanopore Native Barcoding
13. The demonstrator will generate independent DNA sequencing libraries for 16S and ITS samples in the meantime. You will load these libraries in week 5 onto a Nanopore MinION flowcell. Please see associated protocol for details.