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 centrifugal dialysis (12.58 ± 7.15%) and magnetic bead extraction (9.92 ± 3.89%). However, isopropanol precipitation's efficacy was influenced by humic acid concentration in the water matrix. The presence of humic acids significantly affected isopropanol precipitation, with higher humic acid concentrations leading to increased DNA recovery. This suggests that humic acids act as co-precipitators during isopropanol precipitation. We observed that longer DNA fragments (chicken) had lower recovery compared to shorter fragments (salmon) across all extraction methods. Magnetic bead extraction effectively removed inhibitors, while centrifugal dialysis and isopropanol extraction required an additional inhibitor removal step. Isopropanol, magnetic bead extraction, and centrifugal dialysis methods have estimated costs of 7.74, 8.53, and 23.62 USD in 2023 respectively. Overall, isopropanol precipitation was the least expensive and most effective with the highest measured recoveries, but it was dependent on humic acid concentration in the environmental sample.

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 matrix could have increased this DNA recovery. To test how humic acids in the water matrix impact the recovery of DNA with isopropanol precipitation, a fresh salmon-chicken spike was prepared and spiked as described above. The synthetic water matrix was identical as described above but with three treatments of varying humic acid concentrations. The first treatment had no humic acids added, the second had 2.5 mg/L and the third had a 5 mg/L humic acid concentration (Figure 1B). The humic acid in the third treatment was identical to the humic acid concentration used elsewhere in this publication. The DNA was then extracted using an isopropanol precipitation protocol followed by inhibitor removal as described above. Each treatment was processed in triplicate and stored at -20 °C until qPCR analysis.

Figure 1: Experimental design (A) to test the recovery of dissolved eDNA using isopropanol precipitation, magnetic bead extraction, and centrifugal dialysis, and (B) to evaluate the effect of humic acid concentration on the recovery of dissolved DNA using isopropanol precipitation,

Synthetic Water Preparation

To simulate the characteristics of freshwater samples, moderately hard synthetic water was prepared. The synthetic freshwater was composed of the following components: 96 mg/ml NaHCO3, 60 mg/ml CaSO4·2H2O, 60 mg/ml MgSO4, and 4 mg/ml KCl (USEPA, 2002). These concentrations were chosen to represent typical levels found in natural freshwater systems. Humic acids were selected to represent dissolved organic matter (DOM) because it is a known qPCR inhibitor that is co-extracted in many protocols (Green & Field, 2012; Schriewer et al., 2011; Sidstedt et al., 2015, 2020). The humic acids (product number: 53680, Sigma-Aldrich, St. Louis, MO) were first diluted to make a 500 mg/L stock solution and pH adjusted to 8.5 similar to previous experiments (Schefer et al., 2023). This stock was then spiked into the synthetic freshwater to obtain a final concentration of 5 mg/L which is the average DOM value present in freshwater (Thurman, 1985).

DNA spike-in Preparation

To assess the performance of our dissolved DNA extraction methods, we used DNA from two species as spike controls. Sonicated chum salmon (Oncorhynchus keta) DNA (Invitrogen) and genomic chicken (Gallus gallus) DNA were utilized as spike-in materials. Salmon DNA (Invitrogen, Waltham, MA, Cat:15632011), resulting in shorter DNA fragments ranging from 100 to 5000 bp confirmed by measuring with TapeStation (Aligent Tec., Santa Clara, CA) D-5000 kit  (Figure S1). Genomic chicken DNA was extracted from store-bought chicken breasts using a protocol described in Kirtane et al, 2023, as this DNA was the source of longer, high molecular weight with fragments ranging from 6000 to 60,000 bp which was confirmed with TapeStation Genomic DNA kit (Figure S1). 500 µL of DNA from both species (~200 ng/µL each) was combined and vortexed to homogenize the fragment sizes. 20 µL of this stock DNA was spiked into 100 mL of the synthetic water resulting in a final concentration of 0.04 ng/µL of dissolved DNA in each sample.

Isopropanol precipitation

The 100 mL sample was supplemented with 20 mL of 5 M NaCl, followed by the addition of 100 mL of isopropanol. This was then incubated at -20 °C for one hour to facilitate DNA precipitation. After incubation, 50 mL of the sample was added to a 50 mL tube. The 50 mL tubes were then centrifuged at 10,000 x g for one hour to separate the DNA pellet from the supernatant. The supernatant was discarded by pouring it into a waste container, and the same tube was replenished with an additional 50 mL of the sample. This process was repeated iteratively until the entire sample volume was precipitated. Following the final decantation of the supernatant, the DNA pellet was washed twice with 10 mL of 70 % ethanol, each time subjected to centrifugation at 10,000 x g for 30 minutes. The supernatant ethanol was discarded, and the DNA pellet was air-dried completely for 45-60 mins. To re-dissolve the dried DNA pellet, 200 μL of TE buffer warmed to 60 °C was added, and the tube was gently shaken by hand. The dissolved DNA was then transferred into a 2 mL microcentrifuge tube and stored at -20 °C before subsequent analysis.

Magnetic bead extraction

The 100 mL sample was mixed with 80 mL of Environmental Hybridization Buffer (For 500mL: 1 g DTT, 72.5 g NaCl, 125 g PEG 8000, 500 μL 0.5 M EDTA, HPLC water up to 500 mL), followed by the addition of 200 μL of 20 % seramag magnetic beads (SeraMag SpeedBeads, Carboxylate-Modified Magnetic Particles, Hydrophobic, 65152105050250). The solution was thoroughly shaken in a shaking incubator for 45 minutes to ensure proper bead dispersion and binding to the target molecules. Next, 15 mL of the mixture was transferred to a 15 mL tube. The tube was then placed on a 3D-printed magnetic rack, allowing the magnetic beads to migrate toward the magnet. Careful attention was given to removing the remaining solution without disturbing the bead pellet next to the magnet, using a 10 mL serological pipette. Subsequently, the same tube was replenished with an additional 15 mL of the sample solution and placed back on the magnetic rack. This process was repeated iteratively until the entire sample solution had been processed.

After the final removal step, 10 mL of 70 % ethanol was added to the tube. The tube was briefly vortexed to wash the magnetic beads. The Falcon tube was then placed back on the magnetic rack, allowing the magnetic beads to migrate toward the magnet. The ethanol was carefully discarded, and the ethanol wash step was repeated once more. Following the removal of ethanol, the tube was taken off the magnetic rack, and the tube was allowed to air dry for 45 minutes, ensuring complete evaporation of residual ethanol. Subsequently, 200 μL of TE buffer (pre-warmed at 60 °C) was added to the falcon tube. The tube was gently shaken by hand to facilitate the effective elution of the captured molecules. The Falcon tube was then placed back on the magnetic rack, enabling the magnetic beads to migrate toward the magnet. The eluate containing the target molecules was carefully collected to not transfer any magnetic beads, and stored in a 2 mL microcentrifuge tube at -20 °C before subsequent analysis.

Centrifugal dialysis using Amicon® tubes

Of the 100 mL sample, 15 mL was transferred to an Amicon® tube containing the 10 kDa Amicon® Ultra-0.5 Device (Burlington, MA). The Amicon® tube was centrifuged at 5000 xg for 15 minutes. Subsequently, the flow-through was discarded, and the process was repeated iteratively until all 100 mL of the sample had been processed. After the final centrifugation and removal of the flow-through, the filter membrane was gently washed with molecular-grade water using a squeeze bottle. Care was taken to ensure proper rinsing without damaging the filter membrane. Following the wash, a final centrifugation step was performed to ensure the filtrate volume was below the filter membrane. The resulting filtrate, approximately between 200 and 250 μL, was transferred to a 2 mL microcentrifuge tube and stored at -20 °C before subsequent analysis.

Inhibitor removal using ZYMO cleanup kit

Following the dissolved DNA purification, a subsample of 100 μL of the DNA was subjected to a purification step to remove inhibitors using the OneStep PCR Inhibitor Removal Kit (ZYMO, Irvine, CA) per the manufacturer's protocol. Both the inhibitor-removed DNA and the uncleaned aliquots of the DNA extract were stored at -20 °C until further analysis.

Multiplex qPCR

Multiplex qPCR was performed following the protocol outlined in Kirtane et al, 2023, however using only two assays, one for the salmon and one for the chicken. Each qPCR plate used for the analysis also had a 6-point gBlock standard curve ranging from 10² to 10⁷ copies/reaction. qPCR efficiency, the limit of detection (LOD), and the limit of quantification (LOQ) were calculated and reported based on recommended guidelines (Klymus et al., 2020). DNA spikes were also quantified using qPCR. All spikes, samples, and standards were analyzed in triplicate. Each plate consisted of at least six qPCR negative wells.

Data analysis

Percent recovery of the dissolved DNA was calculated using equation 1 where Ce is the concentration of extracted DNA (copies / µL), Ve is the volume of eluate (µL), Cs is the concentration of spiked DNA (copies / µL) and Vs is the volume of DNA spiked (µL).

Equation 1:

We used the Welch Two sample t-test to evaluate whether ZYMO inhibitor removal treatment led to a significant change in the recovery of DNA. One-way ANOVA was performed followed by Tukey’s posthoc to compare the recovery of DNA extracted with the three methods post ZYMO inhibitor cleanup. One-way ANOVA followed by Tukey’s posthoc test was used to evaluate whether the addition of humic acids in the water matrix significantly impacted the DNA recovery rates using isopropanol precipitation. DNA recovery concentrations from both the salmon and chicken DNA were used for the analyses mentioned above. We used t-tests to evaluate whether salmon or chicken DNA, which represent different size classes, had an impact on the recovery of dissolved DNA for each of the three methods. It was ensured that the datasets met the assumptions for the use of parametric tests and the alpha value for significance testing was set at 0.01 for all parametric tests. All assumptions to use parametric statistical tests were met before proceeding with the statistical analyses. All analysis was performed using the ‘stats’ package in base R version 4.3.1 and the graphics were made using the package ‘ggplot2’.

Cost Comparison

The costs for each extraction method were calculated based on the list price without VAT of regents and materials as available in Switzerland with all prices listed in United States Dollars (USD). Laboratory equipment costs such as centrifuges, freezers, etc. are not accounted for along with generic laboratory consumables such as microcentrifuge tubes, gloves, and pipette tips. Nor were labor costs estimated since these differ widely depending on where and by whom the work is carried out.