Dec 13, 2024
Using digital droplet PCR to detect rare Wolbachia endosymbionts
- Alphaxand Njogu1,
- J. Dylan Shropshire1
- 1Department of Biological Sciences, Lehigh University
- Shropshire Lab
Protocol Citation: Alphaxand Njogu, J. Dylan Shropshire 2024. Using digital droplet PCR to detect rare Wolbachia endosymbionts. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l294b3v1y/v1
Manuscript citation:
Njogu, A. K., Logozzo, F., Conner, W. R., & Shropshire, J.D. (2024) Counting rare Wolbachia endosymbionts using digital droplet PCR. bioRxiv. https://doi.org/10.1101/2024.12.10.627731.
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: November 23, 2024
Last Modified: December 13, 2024
Protocol Integer ID: 112667
Keywords: Wolbachia, Drosophila, ddPCR, density, titer, spike-in control, quantifies wolbachia gene copy, copy wolbachia gene ftsz, wolbachia gene copy, absolute wolbachia gene copy number, rare wolbachia endosymbionts this protocol detail, wolbachia strain, quantifying wolbachia endosymbiont, rare wolbachia endosymbiont, wolbachia endosymbionts this protocol, wolbachia density per host cell, wolbachia endosymbiont, versatile ddpcr assay, ddpcr assay, wolbachia dynamics at unprecedented resolution, ddpcr assay measure, sensitive digital droplet polymerase chain reaction, conserved host gene, wolbachia dynamic, using digital droplet pcr, digital droplet pcr, dna purification efficiency, wolbachia density, mid1 assay, ddpcr, gene, drosophila, assay, proprietary dna spike, pcr, wolbachia
Funders Acknowledgements:
Lehigh University Class of '68 Pre-tenure Faculty Award
Grant ID: NA
Abstract
This protocol details three sensitive digital droplet polymerase chain reaction (ddPCR) assays for quantifying Wolbachia endosymbionts. These assays reliably detect as few as 12 Wolbachia gene copies per 20 µL reaction, eliminating the need for sample pooling and preserving valuable individual-level resolution. Each assay targets the single-copy Wolbachia gene ftsZ, using primers and a FAM-labeled probe designed to recognize sequences conserved across 106 supergroup A Wolbachia strains. The singleplex ftsZ-ddPCR assay measures absolute Wolbachia gene copy number per sample, the duplex ftsZ/spike-ddPCR assay quantifies Wolbachia gene copies while correcting for DNA purification efficiency using a proprietary DNA spike-in control, and the duplex ftsZ/mid1-ddPCR assay determines Wolbachia density per host cell by normalizing ftsZ copies to a conserved host gene (mid1). The mid1 assay is designed with homology to 53 Drosophila, Scaptomyza, and Zaprionus species, where the mid1 non-coding region is highly conserved. These sensitive and versatile ddPCR assays will empower researchers to investigate Wolbachia dynamics at unprecedented resolution.
Materials
Materials
- Centrifuge tubes, 1.5 mL, safe-lock, PCR cleanEppendorfCatalog #22363212
- ddPCR Droplet Reader OilBio-Rad LaboratoriesCatalog #1863004
- ddPCR Supermix for Probes (no dUTP)Bio-Rad LaboratoriesCatalog #1863023
- DG8 cartridgeBio-Rad LaboratoriesCatalog #1864007
- DG8 cartridge holderBio-Rad LaboratoriesCatalog #1863051
- DG8 gasketBio-Rad LaboratoriesCatalog #1864007
- Droplet generation oil for probesBio-Rad LaboratoriesCatalog #1863005
- Kimtech Science™ Kimwipes™ Delicate Task WipesKimberly-ClarkCatalog #34155
- Microseal “B” Seal, AdhesiveBio-Rad LaboratoriesCatalog #MSB1001
- PCR plate, 96 wellBio-Rad LaboratoriesCatalog #1863005
- PCR plate, 96 well, ddPCRBio-Rad LaboratoriesCatalog #12001925
- PCR plate heat seal, foil, pierceableBio-Rad LaboratoriesCatalog #1814040
- Pipette tips, filtered, PCR clean and sterile, 0.1 - 10 uLEppendorfCatalog #0030078519
- Pipette tips, filtered, PCR clean and sterile, 2 - 100 uLEppendorfCatalog #0030078543
- Pipette tips, filtered, PCR clean and sterile, 50 - 1,000 uLEppendorfCatalog #0030078578
- Universal DNA Spike ITATAA Biocenter Catalog #DS25SI
- Water, molecular biology gradeMIDSCICatalog #KC248796
Note
Use high-quality plastics throughout this protocol. Low-quality pipette tips, for example, can result in droplet shearing, significantly reducing sample quality.
Oligos
- ftsZ_GroupA_dd forward primer: 5'-GCAGTTAAGGATAGAGCGCC-3'
- ftsZ_GroupA_dd reverse primer: 5'-GGAATGACAATAAGTGTATCCACG-3'
- ftsZ_GroupA_dd FAM-labelled probe: 5'-ACCGTTCGGTTTTGAAGGTGTGCGCCG-3'
- UCE_mid1_dd forward primer: 5'-CATATTGACCTCGGGTTCGG-3'
- UCE_mid1_dd reverse primer: 5'-CGCATTGAATTGGAGTCGC-3'
- UCE_mid1_dd HEX-labelled probe: 5'-TGCAGGCCCGCTTGAGACGCGC-3'
- Universal DNA Spike ITATAA Biocenter Catalog #DS25SI
Note
The ftsZ- and mid1-ddPCR oligos should be used at primer:probe ratio of 900 nM:250 nM.
Spike oligos should be used at the provided concentration.
Equipment
- Centrifuge (Eppendorf, 5420)
- Centrifuge, for plates (Eppendorf, 5430R)
- Droplet generator (Bio-Rad, 1864002)
- Droplet reader (Bio-Rad, 1864003)
- Freezer (any)
- Pipette, 10 μL (Eppendorf, 4861000708)
- Pipette, 100 μL (Eppendorf, 4861000716)
- Pipette, 1000 μL (Eppendorf, 4861000732)
- QX Manager Software
- Rubber Brayer Roller, 4 inch (Amazon, B07YDNKSH6)
- Thermal PCR plate sealer (Bio-Rad, 1814000)
- Thermal Cycler (Bio-Rad, 1841100)
- Vortex mixer (MI0101002, Four E's Scientific)
Protocol materials
ddPCR Supermix for Probes (no dUTP)Bio-Rad LaboratoriesCatalog #1863023
Water, molecular biology gradeMIDSCICatalog #KC248796
DG8 cartridge holderBio-Rad LaboratoriesCatalog #1863051
DG8 cartridgeBio-Rad LaboratoriesCatalog #1864007
Centrifuge tubes, 1.5 mL, safe-lock, PCR cleanEppendorfCatalog #22363212
PCR plate, 96 wellBio-Rad LaboratoriesCatalog #1863005
Droplet generation oil for probesBio-Rad LaboratoriesCatalog #1863005
PCR plate, 96 well, ddPCRBio-Rad LaboratoriesCatalog #12001925
ddPCR Droplet Reader OilBio-Rad LaboratoriesCatalog #1863004
PCR plate heat seal, foil, pierceableBio-Rad LaboratoriesCatalog #1814040
DG8 gasketBio-Rad LaboratoriesCatalog #1864007
Universal DNA Spike ITATAA Biocenter Catalog #DS25SI
Kimtech Science™ Kimwipes™ Delicate Task WipesKimberly-ClarkCatalog #34155
Pipette tips, filtered, PCR clean and sterile, 2 - 100 uLEppendorfCatalog #0030078543
Microseal “B” Seal, AdhesiveBio-Rad LaboratoriesCatalog #MSB1001
Pipette tips, filtered, PCR clean and sterile, 0.1 - 10 uLEppendorfCatalog #0030078519
Pipette tips, filtered, PCR clean and sterile, 50 - 1,000 uLEppendorfCatalog #0030078578
Troubleshooting
Before start
- Collect samples to be analyzed via ddPCR.
- Clean workstation with 10 % (v/v) bleach and 70 % (v/v) ethanol.
Assemble PCR reaction
1h
Remove the following reagents from the freezer and allow to thaw On ice for 00:30:00 :
- ddPCR Supermix for Probes (no dUTP)Bio-Rad LaboratoriesCatalog #1863023
- Water, molecular biology gradeMIDSCICatalog #KC248796
- Primers and probes, as appropriate
- DNA samples
30m
Vortex reagents for 00:00:30 to mix.
30s
Centrifuge at 2000 rcf, Room temperature, 00:00:10 to bring contents to the bottom of each tube.
1m
Select one of the following assays.
Create a master mix in a Centrifuge tubes, 1.5 mL, safe-lock, PCR cleanEppendorfCatalog #22363212 based on the described composition.
The final volume of each assay will be 20 µL , including 2 µL of DNA template.
Calculate a 10% excess.
5m
ftsZ-ddPCR assay reaction composition:
- 10 µL ddPCR Supermix for Probes (no dUTP)Bio-Rad LaboratoriesCatalog #1863023
- 7.5 µL Water, molecular biology gradeMIDSCICatalog #KC248796
- 0.5 µL ftsZ oligo mixture with FAM-labeled probe
- 2 µL DNA template
Note
The ftsZ-ddPCR assay is used to measure Wolbachia titers (absolute abundance).
This assay is useful when:
- Absolute target abundance in the original sample is not a primary concern.
- Technical variations, such as DNA extraction efficiency, are evenly distributed across treatment groups.
- Biological variation between treatment groups is substantial enough to overcome the impact of technical variations.
ftsZ/mid1-ddPCR assay reaction composition:
- 10 µL ddPCR Supermix for Probes (no dUTP)Bio-Rad LaboratoriesCatalog #1863023
- 7 µL Water, molecular biology gradeMIDSCICatalog #KC248796
- 0.5 µL ftsZ oligo mixture with FAM-labeled probe
- 0.5 µL mid1 oligo mixture with HEX-labeled probe
- 2 µL DNA template
Note
The ftsZ/mid1-ddPCR assay is used to measure Wolbachia density (relative abundance).
This assay is useful when:
- The number of Wolbachia cells per host cell is more relevant than the total Wolbachia count.
- Host cell numbers vary between treatment groups or samples.
ftsZ/spike-ddPCR assay reaction composition:
- 10 µL ddPCR Supermix for Probes (no dUTP)Bio-Rad LaboratoriesCatalog #1863023
- 6 µL Water, molecular biology gradeMIDSCICatalog #KC248796
- 0.5 µL ftsZ oligo mixture with FAM-labeled probe
- 1 µL DNA spike-in control primer mixture
- 0.5 µL DNA spike-in control HEX-labeled probe
- 2 µL DNA template
Note
The ftsZ/spike-ddPCR assay is used to measure Wolbachia titer (absolute abundance).
This assay is useful when:
- Technical variations, such as DNA extraction efficiency, can significantly impact results.
- The biological effect size is small and can be masked by technical noise.
- There is a need to back-calculate Wolbachia abundance in the original sample.
Vortex the master mix for 00:00:30 to mix.
30s
Dispense 18 µL of the master mix into each well in a PCR plate, 96 wellBio-Rad LaboratoriesCatalog #1863005 .
10m
Add 2.0 µL DNA template to each reaction.
Add 2.0 µL Water, molecular biology gradeMIDSCICatalog #KC248796 to no template control reactions (NTCs)
10m
Use the Microseal “B” adhesive seal to seal the plate.
Use a roller to ensure the seal is tight.
Mix thoroughly by vortexing the plate for 00:00:10 .
1m
Centrifuge at 2204 rcf, Room temperature, 00:02:00 to bring contents to the bottom of each well.
Note
After centrifugation, check that there are no bubbles across the wells.
If there are bubbles, repeat the centrifugation.
2m
Generate droplets
9m 50s
Insert a DG8 cartridgeBio-Rad LaboratoriesCatalog #1864007 into a DG8 cartridge holderBio-Rad LaboratoriesCatalog #1863051 .
Note
Check that the orientation of the cartridge is aligned properly in the cartridge holder.
Slide the edges of the holder toward the center to snap close.
15s
Peel away the adhesive seal to expose the sample.
Be gentle while removing the seal to avoid aerosolizing the samples.
By pipetting, transfer 19.5 µL of each reaction mix to the sample wells of a DG8 cartridge.
Avoid pipetting air bubbles into the DG8 cartridge.
Note
Check each sample in the DG8 cartridge for air bubbles.
Remove air bubbles by pipetting slowly using a different tip.
Air bubbles can interfere with droplet generation.
1m
By pipetting, add 70 µL Droplet generation oil for probesBio-Rad LaboratoriesCatalog #1863005 to the oil wells in the DG8 cartridgeBio-Rad LaboratoriesCatalog #1864007 .
30s
Insert the DG8 cartridge holderBio-Rad LaboratoriesCatalog #1863051 into the droplet generator.
Close the lid.
Wait approximately00:02:00 for droplets to be generated
2m
While waiting, to prepare the next set of eight reactions.
4m
Open the droplet generator.
Replace the cartridge holder containing droplets with the cartridge holder that was prepared while waiting.
Close the lid to start droplet generation.
Remove the cartridge from the first cartridge holder and check for droplets.
By pipetting, transfer the droplet well (40 µL ) from the DG8 cartridgeBio-Rad LaboratoriesCatalog #1864007 to a clean PCR plate, 96 well, ddPCRBio-Rad LaboratoriesCatalog #12001925 .
Note
If droplet generating is successful, the wells appear opaque.
Pipette slowly to avoid shearing the droplets.
2m
Once all the samples have been transferred, seal the PCR plate with a
PCR plate heat seal, foil, pierceableBio-Rad LaboratoriesCatalog #1814040 using a thermal PCR plate sealer set to heat the foil at 180 °C for 00:00:05 .
Safety information
When using a PX1 plate sealer, ensure the PCR plate is correctly aligned with the heat sealer. Always position the foil with the red line facing upward to prevent it from sticking to the machine and causing damage.
5s
Thermal Cycling
3h 1m
Transfer the sealed PCR plate containing droplets to a thermal cycler.
1m
Run the following program with a ramp rate of 1 °C per 00:00:01 and with a heated lid of 105 °C .
1 cycle of:
- 95 °C for 00:10:00 to activate polymerase and denature DNA.
50 cycles of:
- 94 °C for 00:01:00 to denature DNA.
- 60 °C for 00:02:00 to anneal primers and replicate amplicons.
1 cycle of:
- 98 °C for 00:10:00 to inactivate polymerase.
- 12 °C forever to store amplicons until use.
3h
Read droplets
1h
Transfer the assembled plate to the droplet reader, and secure it with the clamps in the machine.
Check that well A1 is in the top-left position and close the lid.
Safety information
If the plate is not secured properly, the sampling needle can stick to the lid and damage the instrument.
1m
Open the QX Manager software on the attached computer.
1m
If the droplet reader has not been used in the past week, it is important that the system be primed using the QX Manager software.
- Select the "System Utilities" tab.
- Select "Prime."
- Select "Yes" to begin.
- Select "OK" after priming is complete.
2m
Add a plate in the QX Manager software.
5m
Open the Plate Configuration window.
- Select the "Add Plate" tab.
- Select the plus icon beside "Add Plate."
- Select "Configure Plate" to open the plate configuration window with three tabs: "Plate Information," "Well Selection," and "Well Information."
Record general plate information.
- Select the "Plate Information" tab.
- Add a name for the plate and record it in your lab notebook.
- Define the supermix used in the reactions
- Define the file name and save the plate.
Identify the wells in the plate that contain droplets.
- Select the "Well Selection" tab.
- Select the wells that contain droplets.
- Click "Include Selected Wells."
Define the type of experiment and the contents of the wells.
- Select the "Well Information" tab.
- Click "Experiment Type" and select the appropriate type of experiment.
- Enter sample descriptions into the "Sample Description 1" through "Sample Description 4" fields.
- Select the appropriate "Sample Type" for each well.
- Click "Supermix" and select "Supermix for Probes."
- Click "Assay Type" and select "Single target per channel."
- Enter the target (e.g., ftsZ, mid1, spike) in the "Target Name" field.
- Select the appropriate "Target Type" for each target.
- Click "Signal Ch1" and select "FAM."
- Click "Signal Ch2" and select "HEX" if reactions include mid1 or spike and "none" otherwise.
Select "Save" and "Start Run" to begin reading droplets
Note
The software performs a prerequisites check to ensure that the instrument is ready.
This will tell you if ddPCR Droplet Reader OilBio-Rad LaboratoriesCatalog #1863004 needs to be added or if waste needs to be emptied.
Always add 50 mL of 10 % (v/v) bleach to the waste container after emptying.
30m
After the run is complete, save files associated with the run.
- Click "Save" to save the run.
- Select the "Data Analysis" tab.
- Click "Error Model" a select "Poisson" and "95%."
- Select the "Data Table" tab, ensure all columns are reported, and save it as a csv file.
- Select the "Run Information" tab and save the table.
- Select the "Reports" tab, click "Select All," and save the report as a PDF.
- Select the "1D Amplitude," "2D Amplitude," and "Concentration" tabs to save the plots associated with the run.
20m