TITLE: Quantification of fungal ITS Gene Copies Using ddPCR (EvaGreen-based assay: ITS1f-ITS2)
AUTHORS: Eva Petrova, Roey Angel

[DESCRIPTION]
The protocol was designed to quantify microbial eukaryotic fungi using ITS region copy number evaluation by Droplet Digital PCR technology (ddPCR) from Bio-Rad company. 

For the assay, we are using a universal fungal ITS rRNA primer pair:
 
ITS1f         5'- CTT GGT CAT TTA GAG GAA GTA A -3', 38 bp upstream of ITS1 from White et al., 1990
ITS2          5'- GCT GCG TTC TTC ATC GAT GC -3', identical to ITS2 from White et al., 1990until now because of its extreme length variability among the fungi species. In fact, this variability introduced a bias to final copy numbers estimated by qPCR,.

Amplicon size: ~250–600 bp (Bokulich & Mills, 2013; Hoggart et al., 2018)
The same ITS1f and ITS2 primers are recommended for use for fungi identification by the Earth Microbiome Project.
 


[BEFORE_START]
Take all the reagencies out of a freezer and let them temperate to room temperature. 

[GUIDELINES]
The crucial steps that can influence the final assay results a lot are precise pipetting, mixing and dilutions!
Keep in mind that ddPCR technigue do not posses as wide dynamic concentration range as qPCR does. You can easily "overload" the reaction with too much template DNA putting in. In that case you will see only positive droplets at the end and no negative and system will not be able to calculate copy number for them. As a consequence, you usually have to dilute your template DNA more than for qPCR experiments. Ideally fit inside the range of 101 - 104 coppies of target gene. That is why we usually test several dilutions of few samples in advance to see "where we are".
One have to also keep in mind that using ddPCR technology you must work within the format of eight. If you do not fulfil all the wells in a cartridge you still have to put the reagencies inside the empty wells as well. So, think economically before you start.


[STEPS]
SECTION: ddPCR reaction mixture
1. All reagencies must be equilibrated to RT (do not keep them on ice). Mix each of them properly before use. 
SECTION: ddPCR reaction mixture
2. 

 
Prepare the master mix according to the number of samples and mix several seconds by vortexing. Transfer mix into 96-well plate à 20 µl. 
20 µL 

 

 

 

SECTION: ddPCR reaction mixture
3. Add 2 µl of your DNA sample into each well
 2 µL 
SECTION: ddPCR reaction mixture
4.  
Seal the plate (180°C, 5s) with pierceable aluminium foil. 

 
5 s 

SECTION: ddPCR reaction mixture
5. Let the foil cool down and mix the plate vigorously by vortexing 30 s - 1 min.  
30 s 

SECTION: Droplets generation by AutoDG
6. Put all the staff (cartridges, tips, sealed plate with samples and empty 96-well plate in cooling stand) in corresponding amount inside the AutoDG machine (Bio-Rad). 

 
 

SECTION: Droplets generation by AutoDG
7. Make sure there is a right oil bottle (Automated Droplet Generation oil for Probes) connected to the system. 


 
 

SECTION: Droplets generation by AutoDG
8. Choose a position of samples on touch screen and start dropplets generation. Wait after its finished.
 
 
 
 
 

SECTION: Droplets generation by AutoDG
9. Take the plate with droplets out of the machine and seal it with pierceable aluminium foil  (170°C, 3s).

 
3 s 

SECTION: Droplets generation by AutoDG
10. Put immediately the sealed plate into PCR cycler.

 

SECTION: Droplets generation by AutoDG
11. Clean AutoDG machine, waste used consumables.
SECTION: PCR program
12. 1.  95◦C –5′
2.  x 40 {
       a.  95◦C – 30′′
       b.  52◦C – 2′ 
      }
3.  4◦C – 5′
4.  90◦C –5′
5.  10◦C –hold

Set ramp rate for each step to 2°C/sec!

Set reaction volume to 40ul.
40 µL 

 

SECTION: Droplets reading
13. Put the plate into a metal holder, place them together into QX200 reader. 

 
 

SECTION: Droplets reading
14. Set up the QuntaSoft experiment as follows:

Exp. type:        Absolute quantification (ABS)
Supermix:       QX200 ddPCR EvaGreen Supermix 
Target1:          Ch1 (for FAM labeled probes)

Define the position of ech sample.
SECTION: Droplets reading
15. Check the levels of reader Oil and waste - green control (bottles are physically accessible from machines left side). 

 

SECTION: Droplets reading
16. Start measurement.

 

SECTION: Analysis
17. Set up the treshold just above negative control sample in order to distinguish positive (containing PCR products) from negative (do not contain any PCR product) droplets.

 
 
 

SECTION: Analysis
18. Export .csv file with concentrations (copies/ul) that are posessing you the number of copies in 1ul of reaction. To obtain number of copies in 1ul of your input DNA you have to recalculate:

no. of copies in 1ul of input DNA = (concentration value x 22)/ volume of DNA