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Digital droplet Polymerase Chain Reaction (ddPCR)

By: Steen W. Knudsen

For many years now, NIVA has analyzed water samples for the presence of environmental DNA (abbreviated: ‘eDNA’), both in fresh and marine ecosystems. Among other things, NIVA Denmark has completed the MONIS project (‘Monitoring of non-indigenous species in marine waters’ 2014-2023) on behalf of the Danish Environmental Protection Agency.

In continuation of the MONIS project, in May 2022 NIVA Denmark acquired a so-called ‘digital droplet Polymerase Chain Reaction’ machine (ddPCR machine) and had it installed in the laboratory at the University of Copenhagen at the Natural History Museum of Denmark which NIVA Denmark uses to make analyzes of eDNA.

In NIVA Denmark, we have analyzed marine water samples every year since 2017 for the presence of DNA from non-indigenous species. The first results from the analysis of eDNA in water samples using ‘quantitative Polymerase Chain Reaction’ (qPCR) have already been scientifically published and can be read about at (https://doi.org/10.1016/j.scitotenv.2022.153093).

Searching for eDNA in water samples is based on the same PCR technique that is used to search for COVID in swabs from human mucous membranes. But instead of looking for remains of DNA from COVID on cotton swabs, NIVA Denmark is busy looking for remains of DNA from non-indigenous species in Danish waters from hundreds of marine water samples.

If there is eDNA from a non-indigenous species present in a water sample, the PCR technique will ensure that the DNA molecule will be exponentially multiplied, and thus possible to detect. If no eDNA is present in a water sample, no multiplication occurs.

With the ddPCR technique, this multiplication is isolated in thousands of single droplets. The content of each individual droplet can subsequently be counted by a ‘droplet reader’. Each drop thus becomes a technical replicate of the water sample. The number of positive drops can then give insight into where in Danish waters non-indigenous species occur.

As mentioned, NIVA Denmark has since 2017 analyzed water samples for the content of eDNA from non-indigenous species using quantitative Polymerase Chain Reaction (qPCR). However, this method is far more resource-intensive, since three technical replicates quickly cost at least as much as it costs to make a single tube with thousands of drops in a tube per ddPCR.

The machine and method of ddPCR is described here:

Droplet Digital PCR (ddPCR) Technology.

Our goal is to eventually move the analyzes of water samples with more technical replicates from the current qPCR machine, which requires several tubes of extra reactions, to the newly purchased ddPCR machine (Figure 1), which instead makes between 10,000 and 20,000 microscopic drops in the same tube. It helps to make the measurement of eDNA molecules from the non-indigenous species more precise.

From December 2022, it is planned that we in NIVA Denmark will analyze the same marine water samples with both the qPCR and ddPCR methods in order to find out where in the Danish waters non-indigenous species live and how large the occurrences of these species are ( Figures 2 and 3). In the long term, the plan is for all the analyzes to be carried out on the ddPCR machine.

For further information on eDNA and ddPCR: Steen W. Knudsen (steen.knudsen@niva-dk.dk).

Figure 1: The setup with ddPCR involves two machines plus a computer plus a PCR machine. First, more than 10,000 droplets are made with a ‘droplet generator’ (A). Then the plate with the reactions is placed in a PCR machine (not shown in the photograph), where the desired eDNA fragment is copied in millions of copies, each in its own drop in the reaction tube. Then the plate with reaction tubes, each containing thousands of droplets, is brought back and with a computer (B) information about the individual reaction tubes is entered, after which a ‘droplet reader’ (C) determines how many of the thousands of droplets contain the desired eDNA fragment.

Figure 2: Marine water samples from 2021 have already been analyzed with ‘quantitative polymerase chain reaction’ (qPCR) for eDNA fragment from sand mussel (Mya arenaria). The marine water samples from the Danish Environmental Protection Agency each have their own unique sample number that can be compared across analyses. The color code of the collected location indicates how intense the eDNA signal from sand clam is. A white or yellow square can be interpreted as no eDNA or a questionable eDNA signal. Orange, red and black are interpreted as higher concentration of eDNA from the sought species.
Figure 3: The measurements of eDNA in water samples for sand clam can be compared between the two machines. Because the number of technical replicates with ddPCR is over 10,000, against the three technical replicates used for the qPCR analysis, there is now greater precision in the assessments of eDNA in the water samples. The number of eDNA molecules from sand clams is assessed on a logarithmic scale, and is compared here between ddPCR (green) and qPCR (ochre yellow). The gray and white divisions indicate different water samples collected in 2021, while the violet divisions are measurements on positive controls that serve as a reference. Both machines have a lower limit for whether it is possible to quantify the number of eDNA molecules in the extraction from the water sample, which is indicated here by horizontal lines for the two machines (green and ocher yellow). The unique number code of the water samples is reused in this comparison between ddPCR and qPCR, and can thus be found in Figure 2.
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