Materials and Reagents

ddPCR set-up and droplet generation

PCR Plate Heat Seal, foil, pierceable (Bio-Rad, catalog number: 1814040)
DG8^TM Cartridge for QX200^TM/QX100^TM Droplet Generator (Bio-Rad, catalog number: 1864008)
Droplet Generator DG8^TM Gasket (Bio-Rad, catalog number: 1863009)
ddPCR Plates, 96-Well, Semi-Skirted (Bio-Rad, catalog number: 12001925)
QubitTM dsDNA HS Assay kit (ThermoFisher Scientific, catalog number: Q32851)
Restriction endonuclease with a recognition site within the transgene but not between the primer sites (see procedure for details) and compatible digestion buffer
QX200^TM ddPCR^TM EvaGreen Supermix (Bio-Rad, catalog number: 1864033)
QX200 Droplet Generation Oil for EvaGreen^® (Bio-Rad, catalog number: 1864005)

Equipment

ddPCR set-up and droplet generation

Qubit^TM 4 fluorometer (ThermoFisher Scientific, catalog number: Q33238)
QX200^TM droplet generator (Bio-Rad, catalog number: 1864002)
Thermal cycler (e.g., Bio-Rad C1000 Touch thermal cycler, catalog number: 1851148)
Multichannel pipettes (10-100 µl)
Heat sealer (e.g., Eppendorf S100, catalog number: 5391000036)
DG8 cartridge holder (Bio-Rad, catalog number: 1863051)
QX200 droplet reader (Bio-Rad, catalog number: 1864003)
Droplet reader plate holder (Bio-rad, catalog number: 12006834)

Software

Primer design software, e.g., Primer 3 (https://primer3.ut.ee/)
Sequence analysis software, e.g., Benchling (https://benchling.com)
QuantaSoft software (Bio-Rad, catalog number: 1863007)
Software capable of reading comma-separated values (.csv) files (e.g., Microsoft Excel)
Droplet Digital^TM PCR Applications Guide
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6407.pdf

Procedure

In this procedure, transgene copy number is determined by performing two PCR reactions for each sample. The first amplifies a region of the transgene (target gene), and the second amplifies a region of an endogenous gene of known copy number (reference gene). An alternative method is multiplex amplification of the target and reference gene using hydrolysis probes for detection; primers and probes for several plant reference genes have been previously reported (Collier et al., 2017).

Primer design and selection of restriction endonuclease

Use primer design software (e.g., Primer 3) to select primers that amplify a 100-120 base pair region of both the transgene target and reference gene. Primers should optimally have annealing temperatures of ~60°C, similar estimated binding kinetics, and minimal dimerization.

Check that each primer pair efficiently amplifies the desired target in a test PCR reaction. Include control reactions with (i) genomic DNA from a wild-type non-transgenic plant, and (ii) no DNA. Assemble the reaction as shown in Table 1.

Table 1. PCR reaction for testing ddPCR primer pairs

Components	Stock concentration	Final concentration	Volume
2× QX200^TM ddPCR^TM EvaGreen^®Supermix	2×	1×	5 μl
Forward Primer	2 μM	250 nM	1.25 μl
Reverse Primer	2 μM	250 nM	1.25 μl
Genomic DNA			5-50 ng (≤ 2.5 μl)
Nuclease-free Water			Bring reaction volume to 10 μl
Total			10 µl

Incubate the PCR reaction using the cycling conditions recommended by the reagent manufacturer, provided in Table 2.

Table 2. PCR cycling conditions for testing ddPCR primer pairs. Use a 2°C/s ramping rate for all steps.
Step Temperature, °C Time Number of cycles
Enzyme Activation and Thermosetting 95 5 min 1
Denaturation 95 30 s 40
Annealing 60 50 s
Extension 72 50 s
Signal Stabilization 4 5 min 1
90 5 min 1
Hold 4 ∞
1
Visualize the PCR products by agarose gel electrophoresis, ensuring that single clear bands of the expected sizes are obtained for both sets of primers.
Digesting genomic DNA before amplification will ensure that, even if there are two copies of the transgene inserted in tandem, each copy will be on a separate DNA molecule and therefore in a separate droplet. Analyze the transgene sequence for the presence of restriction endonuclease (RE) recognition sequences using sequence analysis software (e.g., Benchling). Select an RE with at least one recognition site within the transgene but no sites between the primers of either the target or reference gene. Avoid the use of restriction endonucleases sensitive to methylation.

ddPCR set-up

Good quality genomic DNA is required for ddPCR. A cetyltrimethylammonium bromide (CTAB)-based method or a commercial plant DNA extraction kit will provide sufficiently pure genomic DNA. Each ddPCR reaction will generally require 5-50 ng genomic DNA.
Notes:
1. To accurately quantitate DNA concentration, we recommend the use of a Qubit fluorometer.
2. The exact quantity of genomic DNA used in each reaction needs to be optimized for the genome size of the organism.
Digest each sample of genomic DNA using the selected RE. An example restriction digestion reaction is shown in Table 3. Different quantities of enzyme and buffer will be required depending on the manufacturer and concentration of the enzyme. Aim to include ~10-30 U enzyme per g genomic DNA.
Note: The EvaGreen^®product notes indicate that digestion of genomic DNA can also be performed by adding 1 μl (2-5 U) desired restriction endonuclease to the PCR reaction.

Table 3. Example reaction for restriction endonuclease digestion of genomic DNA
Components Volume
10× Restriction Enzyme Buffer 1 μl
Restriction Enzyme (10-20 U/μl) 0.5-3 μl
Genomic DNA 200 ng
Nuclease-free Water Bring reaction volume to 10 μl
Total 10 μl
Inactivate the RE by heating. The exact temperature and length of incubation will depend on the specific enzyme and manufacturer. Following inactivation, the digested genomic DNA can be used directly in the next step without further clean-up.

Assemble the PCR reactions in the order shown in Table 4. In addition to the sample reactions, set up control reactions with (i) genomic DNA from a wild-type non-transgenic plant and (ii) no DNA. It is critical that the assembled ddPCR reactions are well mixed prior to proceeding to droplet generation, as every droplet must contain the same concentration of each component.

Table 4. Example ddPCR reaction

Components	Stock concentration	Final concentration	Volume
2× QX200^TM ddPCR^TM EvaGreen^®Supermix	2×	1×	11.25 μl
Forward Primer	2 μM	250 nM	2.8 μl
Reverse Primer	2 μM	250 nM	2.8 μl
Genomic DNA (pre- digested)			5-50 ng (≤ 5.65 μl)
Nuclease-free Water			Bring reaction volume to 22.5 µl
Total			22.5 µl

Note: If multiple samples are being analyzed, as for other PCR methods, it is recommended to make a master mix and dilute concentrated DNA prior to assembling the ddPCR reaction.

Droplet generation
1. Open a DG8^TM Cartridge Holder by pressing the middle latches.
2. Insert the DG8^TM Cartridge into the holder and close the holder by pressing the two halves together (Figure 1).
  
  Figure 1. Workflow of droplet generation. The PCR reactions and oil are added to a cartridge, which is inserted into the holder and held in place by the gasket. Following droplet generation, the emulsified reactions are transferred to a microtiter plate for thermal cycling.
3. Load each 20-µl reaction assembled in the previous step into individual sample wells of a DG8^TM Cartridge using a multichannel pipette.
4. Add 70 μl QX200 Droplet Generation Oil for EvaGreen^® into the oil wells.
5. Cover the cartridge with the DG8^TM Gasket. Loop the holes of the gasket over the four hooks on the cartridge holder to hold the gasket in place.
6. Open the DG8 droplet generator and insert the cartridge holder. Close the lid by pressing the button. The generator will start automatically.
7. Transfer the entire volume of the droplet emulsion (typically 40 μl) to the desired wells of a semi-skirted 96-well ddPCR plate (Figure 1). Cover the plate with a pierceable foil plate heat seal and attach using a plate-sealer according to the manufacturer’s instructions. Rotate the plate and repeat this step to ensure the foil is fully attached.
  Note: To avoid disrupting the emulsified droplets, take care to pipette very slowly. Wide-bore pipette tips (or pipette tips with the extremity removed using a blade) can be used.
8. Place the sealed plate into a thermal cycler and use exactly the same cycling conditions as those used in the test PCR (see Table 2). During the initial heat cycle, the oil surrounding each droplet will thermoset.
Droplet counting
1. When the PCR cycle is complete, switch on the QX200^TM droplet reader and open the QuantaSoft^TM software.
2. In the QuantaSoft^TM software, start a new experiment (Template > New).
3. Select the wells to be detected and enter the sample names or record the well positions for target and reference genes for each sample.
4. Select the key parameters in the well editor as follows:
  1. Experiment: ABS (absolute quantitation).
    Note: The QuantaSoft^TM software provides three default experiments: Absolute Quantitation (ABS), Rare Event Detection (RED), and Copy Number Variation (CNV). Both ABS and CNV can be used for copy number determination. For CNV mode, a known reference gene copy number needs to be provided. The ABS mode is more flexible. For example, the copy number of the reference gene is not required, and the relative ratio of target and reference can be calculated.
  2. Supermix: QX200^TM ddPCR EvaGreen Supermix.
  3. Target 1 type: Ch1 Unknown.
  4. Target 2 type: Ch2 Unknown.
  5. Name: Enter sample names, e.g., transgenic line x, reference gene. This can help to distinguish which wells are for transgene and which are for reference genes.
5. Apply the chosen parameters to the selected wells by clicking ‘OK’.
6. Remove the PCR plate from the thermocycler and insert into the base of a plate-reader holder. Cover the plate with the lid and press the latches to lock the holder.
7. Open the QX200^TM droplet reader, insert the plate-reader holder, and close the lid.
8. Check that the indicator lights for ‘power’, ‘bottle levels’, and ‘plate in place’ are green.
9. Click ‘Run’ in the QuantaSoft^TM software. Select EvaGreen in the ‘Run Option’ dialog. Click OK to start the reading.

Data analysis

When reading is complete, click on ‘Analysis’.
Click the ‘1D amplitude’ button to view the distribution of positive and negative droplets. An example distribution is shown in Figure 2.

Figure 2. Example plot showing positive (blue) and negative (grey) droplets for eight samples (A01-H01). The X-axis (event number) indicates the number of droplets measured across the total experiment, and the Y-axis shows the amplitude of fluorescence.

Note: A single reaction should produce 12,000-20,000 droplets. The calculation of copy number relies on the ratio of positive to negative droplets. If the positive droplets saturate (most droplets are positive), this means that every droplet had the target molecule and it is likely that either the primers are non-specific or too much DNA was used in the reaction. To obtain an accurate result, primer specificity should be checked and/or the reactions repeated with less DNA.
The threshold, which distinguishes positive droplets from negative droplets, as well as the concentration value, is automatically determined; however, if desired, the threshold can be manually adjusted.
Click the “Concentration” button. Export the concentration data as a comma-separated values (.csv) file.
Open the .csv file in the appropriate software (e.g., Microsoft Excel). Any sample names provided in the QuantaSoft^TM software will be in the ‘Sample’ column. The ‘Concentration’ column contains the number of PCR amplicons per μl required to calculate copy number.

The copy number of the target gene is calculated by (C_target/C_ref) * N_ref, where C_target is the concentration of the transgene, C_ref is the concentration of the endogenous gene, and N_ref is the copy number of the reference gene. In the example provided in Table 5, the reference gene is a single copy homozygous gene in the Arabidopsis genome; thus, there are two copies in each of the diploid somatic cells from which DNA was extracted. In line 1, the concentration of the target (C_target) is 115 copies/μl, and the concentration of the reference is (C_ref) is 227 copies/μl. Therefore, the copy number of the transgene is (115/227)*2 = 1.0, meaning that line 1 has a single copy of the transgene (hemizygous). In line 2, C_target= 784 copies/μl and C_ref = 173 copies/μl. Therefore, the copy number of the transgene is (784/173)*2 = 9.1, meaning that line 2 has nine copies of the transgene. In line 3, C_target= 11.7 copies/μl and C_ref = 333 copies/μl. Therefore, the copy number of the target transgene is (11.7/333)*2 = 0.07 copies, which means that line 3 is unlikely to contain a transgene.

Note: When determining transgene copy numbers in polyploid plants, many endogenous genes typically presenting as a single-copy gene may be present on multiple genomes. For example, there may be four copies of a typical single-copy reference gene in the diploid cells of a tetraploid plant; therefore, the concentration value for a single-copy hemizygous transgene will be one quarter of the reference gene. The method described above can also be used for determining transgene zygosity in progeny generations; however, since the ddPCR protocol will only provide raw values, knowledge of the copy number of the parent line(s) will be required. For example, if the diploid cells of a progeny plant are estimated to contain six copies, knowledge of the copy number of any parental line(s) is required to determine whether the plant has six independent hemizygous loci or three homozygous loci.

Table 5. Example ddPCR data from three transgenic plants (Lines 1-3) showing the number of positive and negative droplets obtained in which the transgene (target) and a known-copy endogenous gene (ref) were amplified. The column showing the concentration (number of PCR amplicons per μl) is used to calculate the copy number.

Well	ExptType	Sample	Status	Concentration	Supermix	Positives	Negatives
A01	Absolute Quantitation	Line 1 target	OK	115	QX200 ddPCR EvaGreen Supermix	1648	16017
A02	Absolute Quantitation	Line 1 ref	OK	227	QX200 ddPCR EvaGreen Supermix	3283	15412
B01	Absolute Quantitation	Line 2 target	OK	784	QX200 ddPCR EvaGreen Supermix	8398	8869
B02	Absolute Quantitation	Line 2 ref	OK	173	QX200 ddPCR EvaGreen Supermix	2321	14615
C01	Absolute Quantitation	Line 3 target	OK	11.7	QX200 ddPCR EvaGreen Supermix	113	11313
C02	Absolute Quantitation	Line 3 ref	OK	333	QX200 ddPCR EvaGreen Supermix	3061	9346

Acknowledgments

We thank Oleg Raitskin for technical help and Simon Foster for equipment maintenance. We acknowledge financial support from the UK Biotechnology and Biological Sciences and the Engineering and Physics Research Councils (BBSRC and EPSRC) synthetic biology for growth program (OpenPlant Synthetic Biology Research Centre BB/L014130/1), as well as grants BBS/E/T/000PR9816 and BB/R021554/1. This protocol was developed to generate the transgene copy number data presented in Cai (2020).

Competing interests

No competing interests to declare.

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