DOI: 10.5061/dryad.z08kprrnk
title: Improving the recovery for dissolved eDNA State: A comparative analysis of isopropanol precipitation, magnetic bead extraction, and centrifugal dialysis

description: 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.
description: <strong>Experimental setup</strong>
 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, <strong>Synthetic Water
 Preparation</strong> 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). <strong>DNA spike-in
 Preparation</strong> To assess the performance of
 our dissolved DNA extraction methods, we used DNA from two species as
 spike controls. Sonicated chum salmon (<em>Oncorhynchus
 keta</em>) DNA (Invitrogen) and genomic chicken (<em>Gallus
 gallus</em>) 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.
 <strong>Isopropanol precipitation </strong>
 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.
 <strong>Magnetic bead extraction </strong>
 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.
 <strong>Centrifugal dialysis</strong>
 <strong>using Amicon® tubes</strong> 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.
 <strong>Inhibitor removal using ZYMO cleanup kit
 </strong> 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.
 <strong>Multiplex qPCR </strong>
 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 10<sup>2</sup> to
 10<sup>7</sup> 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. <strong>Data analysis
 </strong> Percent recovery of the dissolved DNA
 was calculated using equation 1 where <em>Ce</em> is the
 concentration of extracted DNA (copies / µL), <em>Ve</em> is
 the volume of eluate (µL), <em>Cs</em> is the concentration of
 spiked DNA (copies / µL) and <em>Vs</em> 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’. <strong>Cost Comparison
 </strong> 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.
description: # 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 precipitation A = Amicon centrifugal dialysis  6\) Inhibitor: Shows what inhibitor removal method was used. N = None, Z = Zymo ## Sharing/Access information There are no links to other publicly accessible locations of the data: