Oct 01, 2024

Public workspaceCopy number variation analysis by ddPCR

DOI
dx.doi.org/10.17504/protocols.io.q26g7pkd8gwz/v1
  • 1University of California, San Francisco
Katie Jing Kay Lam
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DOI: dx.doi.org/10.17504/protocols.io.q26g7pkd8gwz/v1
External link: https://clellandlab.ucsf.edu/protocols
Protocol Citation: Katie Jing Kay Lam, Olubankole Aladesuyi Arogundade, Claire D Clelland 2024. Copy number variation analysis by ddPCR. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7pkd8gwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 25, 2024
Last Modified: January 25, 2024
Protocol Integer ID: 94179
Keywords: Bio-Rad, ddPCR
Abstract
This protocol describes copy number variation analysis using Bio-Rad Droplet Digital PCR ddPCR and QuantaSoft Software (with modifications).
Materials
ABC
ReagentManufacturerCatalog No.
ddPCR Copy Number Assay: RPP30, Human, Homo sapiensBio-RaddHsaCP1000485
ddPCR Supermix for Probes (No dUTP)Bio-Rad1863024
Droplet Generation Oil for ProbesBio-Rad1863005
ABC
Equipment/ConsumableManufacturerCatalog No.
C1000 Touch Thermal Cycler with 96–Deep Well Reaction ModuleBio-Rad1851197
ddPCR 96-Well PlatesBio-Rad12001925
DG8 CartridgesBio-Rad1864008
DG8 Cartridge HolderBio-Rad1863051
DG8 GasketsBio-Rad1863009
PCR Plate Heat Seal, foil, pierceableBio-Rad1814040
PX1 PCR Plate SealerBio-Rad1814000
QX200 Droplet Digital PCR SystemBio-Rad
ddPCR workflow
ddPCR workflow


ddPCR reaction set up
ddPCR reaction set up
Thaw all components to room temperature
Prepare 20X target primers/probe
AB
Forward primer (100 µM)18 µL
Reverse primer (100 µM)18 µL
Target probe (FAM) *light sensitive5 µL
RNase/DNase-free H2O59 µL
Note: Store at -20°C
Prepare Master Mix – Number of reactions + 1 (as extra)
AB
2X ddPCR Supermix for Probes (No dUTPs)11 µL
20X target primers/probe (FAM)1.1 µL
20X reference primers/probe (HEX/VIC)1.1 µL
RNase/DNase-free H2OUp to 22 µL (including DNA sample)
Note: RRP30 is used as the reference primer/probe in this protocol
For each PCR tube: DNA sample (Up to 350 ng) + Master Mix = 22 µL
Droplet generation
Droplet generation
Insert DG8‱ Cartridge into cartridge holder



FIRST - Load >20µL sample into wells
Note:
- Add a 50:50 mix of Supermix & H2O in empty wells
- All 8 wells must be loaded with samples or Supermix + H2O
- Make sure there are no bubbles
Critical
Load 70 µL of generation oil into all 8 wells
Note:
- Make sure samples are loaded before generation oil
- Generation oil is one-time use/can only be used within a day once opened
Apply a DG8 Gasket on cartridge
Load cartridge into QX200‱ Droplet Generator




Transfer Droplets
Transfer Droplets
Droplets should be transferred to 96-well plate within 5 mins of generation
Critical
Transfer 40 µL droplets to a ddPCR 96-Well Plate
Note: Pipette SLOWLY for droplet suction and dispensing
Cover 96w plate with a foil seal sheet
Note: The side with red line should be facing up



Seal plate in a PX1 PCR Plate Sealer at 180°C for 5 secs



ddPCR cycle
ddPCR cycle
ddPCR cycle should be started within 40 mins after sealing
Critical
Place plate into Bio-Rad Thermal Cycler with 96-Deep Well C1000 block

ABCD
Step Temperature Time
Enzyme Activation94°C10 minsRamp rate
Denaturation94°C30 sec2°C/sec
Annealing/Extension*50-65°C1 min
Repeat from step 2 (39x)
Enzyme Deativation98°C10 mins
Hold4°C∞
Note: Optimized annealing temperature when using primers/probe set for the first time by using gradient temperatures
PCR
Reading the plate
Reading the plate
Set plate in room temperature for 1-2mins
Note: The plate can be stored at 4°C for ≤3 days before reading
Place plate in Bio-Rad QX200‱ Droplet Reader



Open QuantaSoft Software & set up


Select a well
E.g. Sample
- Enter sample name
- Experiment: CNV2
- Supermix: ddPCR Supermix for Probes (no dUTP)
E.g. Target 1 (FAM)
- Enter name
- Type: Ch 1 Unknown
E.g. Target 2 (HEX/VIC)
- Enter name (RPP30)
- Type: Ch 2 Reference
Repeat (copy & paste) for every well used in the plate
Save template & RUN
Data analysis
Data analysis
Check if there are >10k events for each sample
Set threshold manually (pink line) - if necessary
Note: Make sure the positive droplets (blue/green) are separated from the negative droplets (grey)

Analyze
Protocol references
1. Figure 4, and part of Figures 1, 7 are created with BioRender.com
2. Bio-Rad. Droplet Digital‱ PCR Applications Guide. Retrieved May 25, 2023, from https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6407.pdf
3. Bio-Rad. QX200‱ Droplet Generator Instruction Manual. Retrieved May 25, 2023, from https://www.bio-rad.com/webroot/web/pdf/lsr/literature/10031907.pdf
4. Bio-Rad. PX1‱ PCR Plate Sealer Instruction Manual. Retrieved May 25, 2023, from https://www.bio-rad.com/sites/default/files/webroot/web/pdf/lsr/literature/bulletin_10023997.pdf
5. Bio-Rad. QX200 Droplet Reader and QuantaSoft Software Instruction Manual. Retrieved May 25, 2023, from https://www.bio-rad.com/webroot/web/pdf/lsr/literature/10031906.pdf
6. Data from Figures 7 are generated from Bio-Rad QuantaSoft‱ Analysis Pro Software