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Droplet digital PCR data from method testing to improve eDNA yield and reduce inhibitors from environmental water samples

April 9, 2019

Data files for manuscript "Improving eDNA yield and inhibitor reduction through increased water volumes and modified multi-filter isolation techniques". These include the results of different treatments for inhibitor removal from a water sample and DNA yield quantities from such tests.

Environmental DNA (eDNA) methods detect genetic material that is shed into the environment through skin cells and excrement by plants and animals. These methods are increasingly being applied to detect imperiled and invasive species for range assessment and occurrence estimates. To improve the recovery and detection of eDNA, we tested and optimized three basic protocols to 1) assess whether eDNA concentration increase in a linear fashion, 2) test the effectiveness of using multiple filters to allow for DNA isolation from larger volumes of water and reduce clogging, and 3) reduce the amount of inhibitory compounds retained during DNA isolation. Environmental DNA was found to increase substantially, possibly indicating difficulty in pelleting or binding to tube walls when using pure water and cell-free DNA. In our second experiment we wanted to ameliorate the issue of filters clogging from particulates and organic material, which can limit the volume of filtered water, and in turn the amount of DNA collected and extracted. We developed a scaled-up Phenol-Chloroform-Isoamyl (PCI) procedure in 5 mL tubes that allows for up to five filters to be combined in a single DNA isolation, resulting in larger volumes of water to be filtered rapidly. We found that increasing the filtered volume resulted in 1.4 times the yield of target DNA. In certain water samples, inhibition from organic material in the environment can reduce or eliminate eDNA detection in a PCR-based assay. In our final experiment, we tested three isolation methods to remove inhibitors and allow for maximum recovery of eDNA. The use of CTAB as a short-term storage buffer followed by a PCI isolation within 5-8 days provided the highest eDNA yields, without the need for an inhibitor removal kit. In summary, we've shown precise estimation of eDNA, especially at higher concentrations. We have also identified a way to increase filtering volumes and reduce inhibition for improved detection of eDNA.