TITLE: Quantification of 16S rRNA Gene Copies Using ddPCR (EvaGreen-based assay: 338F-805R)
AUTHORS: Roey Angel, Eva Petrova

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes how to quantify 16S rRNA bacterial gene or transcript copy numbers using </span><a href="https://www.bio-rad.com/en-cz/applications-technologies/droplet-digital-pcr-ddpcr-technology?ID=MDV31M4VY" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Droplet Digital PCR technology (ddPCR)</span></a><span> from Bio-Rad. This is an up-to-date modification of a classical bacterial enumeration qPCR-assay. This assay uses the EvaGreen™ chemistry. The primers are taken from </span><a href="http://dx.doi.org/10.1002/bit.20347" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Yu et al. (2005)</span></a><span>.</span></div></div>

[STEPS]
?. [Primers]
The assay uses the following universal 16S bacteria primers:  ABCD1NameDirectionSequenceTarget region12BAC338FFACT CCT ACG GGA GGC AG338-3543BAC805RRGAC TAC CAG GGT ATC TAA TC785-805     1. Relative to E. coli SSU rRNA gene
ABCD1NameDirectionSequenceTarget region12BAC338FFACT CCT ACG GGA GGC AG338-3543BAC805RRGAC TAC CAG GGT ATC TAA TC785-805
?. [PCR mixture]
All reagents must be equilibrated to   (do not keep them on ice). Mix each of them properly before use.
0 Room temperature
?. [PCR mixture]
ABCD1ReagentFinal conc.1 tube (22 μl)plate (22 μl x 100)2PCR H2O&nbsp;8.68603QX200 ddPCR EvaGreen Supermix1x1111004BAC 338F (10 μM)0.1 μM0.2205BAC 805R (10 μM)0.1 μM0.2206Template&nbsp;22 x 100Prepare the master mix according to the number of samples (incl. at least one NTC sample) and mix for several seconds by vortexing or pipetting. Transfer mix into 96-well plate à .
ABCD1ReagentFinal conc.1 tube (22 μl)plate (22 μl x 100)2PCR H2O&nbsp;8.68603QX200 ddPCR EvaGreen Supermix1x1111004BAC 338F (10 μM)0.1 μM0.2205BAC 805R (10 μM)0.1 μM0.2206Template&nbsp;22 x 100
20 µl
Tip: use a mechanical or electronic dispenser (e.g. Multipette, Pipettman, or a multichannel pipette) during this step to speed up the work.
?. [PCR mixture]
Add  into each well.
[DNA template]
?. [PCR mixture]
Seal the plate ( ) with a pierceable aluminium foil.
180 °C
?. [PCR mixture]
Let the foil cool down and mix the plate vigorously by vortexing for  -
?. [Droplet generation using AutoDG]
Place the cartridges, tips, sealed plate with samples and an empty 96-well plate into their appropriate positions in the  QX200 AutoDG Droplet Digital PCR System (Bio-Rad).
Note: Two pipette tips are needed for each sample!
?. [Droplet generation using AutoDG]
Make sure that the correct oil bottle—Automated Droplet Generation oil for EvaGreen—is connected to the system.
The droplet generation oil is prone to expire about one year after opening. Replace the oil if it has turned milky.
?. [Droplet generation using AutoDG]
Mark the position of the samples in the plate on the touch screen and press "START" initiate the droplet generation.
After the droplet generation is finished, the system will you automatically display a message about the success or failure of the procedure. Nevertheless, even after a successful run, it is advisable to inspect the wells and ensure that two separated phases are clearly visible. Upper part with droplets and lower clear oil phase.
?. [Droplet generation using AutoDG]
Take the plate with droplets out of the AutoDG and seal it with pierceable aluminium foil  (, ).
170 °C
?. [Droplet generation using AutoDG]
Immediately place the sealed plate into PCR cycler (see below) and initiate the reaction.
The droplets are unstable at this stage. Proceed to the next step as soon as possible. Following PCR, the droplets become stable and can be kept at    for some time (up to 24 hours) before measurement.
?. [Droplet generation using AutoDG]
Clean the AutoDG and discard used consumables.
?. [PCR program]
1.  2.  x 5 {       a.         b.  ′ ( each step)      }3.  x 35 {       a.         b.       }4.   5.   6.    holdSettings:Set the reaction volume to  .
95 °C
95 °C
60 °C
-1 °C
95 °C
55 °C
4 °C
90 °C
10 °C
40 µl
After the run is finished check if there are still two phases present.Let the plate cool down before downstream measurement To maximise the droplet count, leave the plate overnight in a fridge before processing the samples in the QX200 Droplet Reader. This is because the droplets tend to stick together after the PCR step, but loose this tendency after prolonged co
?. [Droplet reading]
Put the plate into a metal holder, place them together into QX200 Droplet Reader.
Switch on the reader 30 min before measurement.
?. [Droplet reading]
Set up the QuntaSoft experiment as follows:  AB1Exp. typeAbsolute quantification (ABS)2SupermixEvaGreen ddPCR Supermix3Target1Ch1Define the position of each sample.
AB1Exp. typeAbsolute quantification (ABS)2SupermixEvaGreen ddPCR Supermix3Target1Ch1
?. [Droplet reading]
Check the levels of reader Oil and waste - green control (bottles are physically accessible from the left side of the device).
If the instrument was not in use for longer than one week, it has to be primed first (oil flushed).
?. [Droplet reading]
Start the measurement.
After the count is finished go over the results and check how many droplets were counted for each sample. To get a reliable count the number of droplet should be above 12.000. On average, droplet counts range from 16.000 to 18.000.
?. [Analysis]
Set the threshold just above the negative control sample in order to distinguish positive (droplets containing PCR products) from negative (empty) droplets.
QuantaSoft will automatically calculate a copy number of the target gene for each sample using its Poisson distribution algorithm. For these calculations, a certain portion for the droplets must be negative. If the sample contains only positive droplets it cannot be evaluated properly (see an example below).
?. [Analysis]
Export a CSV file with concentrations (copies µl-1). To obtain the number of copies in 1 µl of template DNA use the following formula:no. of copies in 1 µl of template DNA = ddPCR conc. x 22 / volume of template DNA