we created a duplex ddPCR containing two FAM probes of different mutations and one HEX probe for one of these two mutations. when analyzing the results, two mutant clusters were shown in the 2D plot, However, also two clusters were formed for the empty droplets and for the wild type droplets. The two clusters are right above each other. Does anyone know why this occurs? is it because the probes compete with each other?
Hi Raisa,
Can you please give more details of your experiments? You said it was duplex, but from what you described it seems like triplex. What were the primer/probe concentrations for each of the FAM assays. What was the sample? Is this the droplet readout of only one well?
the sample includes artificial DNA. The probes for two mutations are added. for mutation one the WT and MT primers and probes are added (0.5 uM primer and 0.2 uM probe) for mutation two only the MT primers and probes are added (0.45 uM primer and 0.25 uM probe).
I repeated this test about four times (on different days) and still every time there are two empty and WT clusters in de 2D plots.
Do the droplets which are located in the upper layer contain more probe than those in the lower layer?
If I understood correctly, you are targeting WT, mutation 1 (M1) and mutation 2 (M2). in such case, your output seems ok, since you have several possible combinations of targets in individual droplets. I've tried to summarize it in the attached image.
I would suggest using Quantasoft Analysis Pro for the analysis of multiplex results, as it enables selection of all possible clusters, depending on the degree of multiplexing.
Hi Raisa, The image sent through by David looks like a good interpretation. The Quantasoft pro software allows you to put multiple thresholds (rather than the simple cross hair) - you can download this off the biorad website. I would also do a dilution series of your template as you can predict how many droplets you would expect in each of the clusters (red ovals that David has put in the image) for each dilution and check it against waht you observed. There is also a "missing" droplet cluster with the M2+WT molecules in your figure. This might be subsumed into one of the other clusters (due to competition - see next section) or the differences in concentration of the three molecules may mean that the probability of getting this cluster is low. It may appear in the dilution series.
The other question I have is if I understand your description of the primers and probes correctly then you are detecting SNVs and so have common primers for the different targets that are specified by the probes? For this you will start to see competition in the droplets when the sample concentration gets higher. I can already see the droplet cluster that contains M+M2+WT that is dropping down in amplitude in both channels. You can draw a diaganol line from the M1+M2 cluster down to the WT cluster that cuts through the M1+M2+WT and M1+WT clusters that indicates this. This isn't a problem (you can't do anything about it even if it is) but it might make setting the thresholds a bit harder. If possible I would use the cross-hairs and not the box, circle or lasso tools as it is almost impossible to repeat their positions from one experiment to another. If you don't know what I mean by competition then I have a section in a paper a few years ago (2016; BDQ Fundamentals of mulitplexing with dPCR) as this is a pet project of mine!
Hope that helps and good luck!
Hi Raisa
An Explanation may be crosstalk. We had some crosstalk (from Hex into Fam) and could reduce it by reducing the concentration of the Hex-Probe. Of Course different Alleles may also be an Explanation. However, reducing the probe concentration wouldn't have any Impact in this case.
Success
René
- Badges
- Science topic