Improving the recovery for dissolved eDNA State: A comparative analysis of isopropanol precipitation, magnetic bead extraction, and centrifugal dialysis

Environmental DNA consists of multiple states including but not limited to membrane-bound, adsorbed, and dissolved states. Because of their chemical and physical properties, each of these states may have different degradation and transport potential. Essential to the study of eDNA states is being able to isolate them from an environmental sample. Here we focus on improving the DNA recovery of the dissolved state of eDNA from water. We compared three recovery methods, namely isopropanol precipitation, magnetic bead extraction, and centrifugal dialysis. We evaluated the effectiveness of these methods based on the measured DNA recovery of two different species’ DNA of different sizes. DNA recovery was assessed with qPCR. We also checked for the effect of inhibitor removal, and lastly estimated the cost of reagents and consumables (not labor). The DNA recovery varied among extraction methods, with isopropanol precipitation yielding the highest recovery (52.47 ± 19.69%), followed by centrifu..., Experimental setup
The experimental setup consisted of processing 100 mL of synthetic water samples through three DNA concentration/extraction methods, isopropanol precipitation, magnetic bead extraction, and centrifugal dialysis (Figure 1A). The 100 mL of synthetic water was spiked with target DNA from two species with two different non-overlapping strand sizes (Figure 1). Each method treatment consisted of five replicates and one negative control with no spiked DNA. Post-treatment, a partial volume of the DNA extract from all treatments was passed through an inhibitor removal step to test the effectiveness of each extraction method on inhibitor removal. All samples, spikes, and negative controls were analyzed using a multiplexed qPCR to quantify the recovered concentration of spiked DNA.
Results of the experiment described above showed a dramatic increase in recovery compared to previous studies (eg: Kirtane et al., 2023). We hypothesized that humic acids added to the synthetic water ..., , # Improving the Recovery for Dissolved eDNA State: A Comparative Analysis of Isopropanol Precipitation, Magnetic Bead Extraction, and Centrifugal Dialysis

[https://doi.org/10.5061/dryad.z08kprrnk](https://doi.org/10.5061/dryad.z08kprrnk)

## Description of the data and file structure

This dataset contains qPCR data from an experiment to evaluate methods for extracting eDNA from its dissolved state

This dataset contains one excel file \"dissolved state data.xlsx\" with the following columns 

1\) Position: This indicated the position on the qPCR plate

2\) ID: This indicated the sample identification. Please refer to the methods section for accurate interpretation of these values 

3\) ct: This value indicated the ct value expired from the qPCR instrument. Black or NA cells indicate no qPCR amplification of DNA  

4\) Probe: Indicated what qPCR probe was used for the given measurement 

5\) Method: shows the method of extraction. N = None, M = magnetic bead, I = isopropanol precipitatio...