Wizard® Enviro TNA Kit: Efficient Extraction of Environmental DNA (eDNA) from River Water

In this study, a total of eight river water samples were collected from the Hudson River by the New York Department of Environmental Conservation (NYDEC) to perform environmental DNA (eDNA) extraction and analysis. The samples were collected in two sets of four, with each set containing samples at various volumes: 50ml, 200ml, 500ml and 1L.

One set of samples was filtered in the field through 50mm 1.5µm Cytiva Whatman™ Binder-Free Glass Microfiber filters, Grade 934-AH Circles, while the second set was sent on ice to The Center for Functional Genomics (CFG). Upon receipt, all samples were immediately frozen and stored at –20°C until extraction, which took place within a two-week period.

Filtered samples were purified with the Qiagen DNeasy Blood & Tissue protocol, while the remaining water samples at 50ml, 200ml and 500ml were purified using the Promega Wizard® Enviro TNA protocol. The 1L sample was not processed due to insufficient kit reagents.

The performance of the eDNA extraction kits was evaluated based on the following parameters:

• Total Nucleic Acid (TNA) recovered (quantified in nanograms for each sample).
• Extraction filter capacity (TNA capacity for each sample volume). 
• eDNA sample purity (assessed through A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ values using a NanoDrop® UV spectrophotometer).
• Targeted eDNA yield (kit recovery of species-specific eDNA assessed through quantitative PCR [qPCR]).

DNeasy® Blood and Tissue Method for eDNA Extraction from Filter Samples

1. Sample filters were thawed at room temperature, opened with sterile forceps and homogenized before returning to 5ml sample collection tubes. Forceps were decontaminated with DNA Away™ solution (Thermo Fisher) and 80% ethanol between each use.
2. To each filter in the tube 500µl of Qiagen Buffer ATL supplemented with 55.6µl of Qiagen Proteinase K was added. Samples were vortexed to mix, then incubated at 56°C for 3 hours with regular agitation (vortexed for 10 seconds every 15 minutes) or 4°C overnight to lyse.
3. Next, 10µl of Qiagen RNase A (100mg/ml) was added to each sample, vortexed to mix, and then incubated at RT for 5 minutes.
4. To each sample, 550µl of Qiagen Buffer AL was added. Samples were vortexed to mix, followed by the addition of 550µl of 100% ethanol and vortexed again to mix.
5. Sample tubes were centrifuged at 1,800 × g for 2 minutes to pellet debris.
6. Using a wide-bore P1000 tip, clarified supernatants were transferred to 2ml collection tubes.
7. Next, 500µl of supernatant was transferred to DNeasy® Mini spin columns in 2ml collection tubes. Samples were centrifuged at 6,000 × g for 1 minute, flowthroughs discarded, and this step was repeated for all remaining samples.
8. Columns were transferred to new 2ml collection tubes, then 500µl of Qiagen Buffer AW1 was added to each column, followed by centrifugation at 6,000 × g for 1 minute, discarding flowthroughs.
9. Columns were transferred to new 2ml collection tubes, then 500µl of Qiagen Buffer AW2 was added to each column, followed by centrifugation at max speed (16,873 × g) for 5 minutes to dry column membranes, discarding flowthroughs.
10. Columns were transferred to fresh 1.5ml Eppendorf tubes, then 200µl of Qiagen Buffer AE was applied directly to each membrane, incubated at room temperature for 5 minutes then centrifuged at maximum speed for 1 minute.
11. For each sample, 200µl eluate was reapplied to membranes, incubated for 5 minutes at room temperature, and then centrifuged again at maximum speed for 3 minutes to maximize eDNA recovery.

Promega Wizard® Enviro TNA Method for eDNA Extraction from Water Samples

Three river water samples (50ml, 200ml and 500ml) were processed following the Promega Wizard® Enviro TNA protocol as per manufacturer’s instructions, with the following modifications due to larger sample volumes:

• Each sample was split into multiple 20ml aliquots then processed following the standard protocol, scaling all kit reagents accordingly.
• All aliquots of the same sample were processed through the same individual Promega PureYield™ Binding Column provided in the kit (a total of three columns, one for each sample, was used for processing).
• Final drying of column membrane was extended to 2 minutes to ensure complete evaporation of ethanol from column membrane (see Step 7 of Total Nucleic Acid Extraction and Clean-Up in the Wizard® Enviro Total Nucleic Acid Kit Technical Manual, #TM662).
• Column incubation with 20µl of nuclease-free water at 60°C for elution was extended to 5 minutes at room temperature to be equivalent to the DNeasy® protocol. Eluates were centrifuged at 10,000 rpm for 2 minutes. This step was repeated for a total of two 40µl elutions for each sample.

The analysis of the eDNA samples was conducted to evaluate nucleic acid recovery and identify any carryover contaminants using UV spectrophotometry. The NanoDrop® UV spectrophotometry revealed that the filter samples processed through the DNeasy® Blood & Tissue Kit yielded a range of approximately 400ng (from the 50ml sample) to 2,200ng (from the 1L sample) per filter. It was observed that lower filtered volumes resulted in proportionately less nucleic acid.

On the other hand, the water samples extracted via the Promega Wizard® Enviro TNA Kit exhibited consistently higher yields across all volumes extracted, with a range of about 2.4–3µg of total nucleic acid for all samples. This represents an increase of up to five times higher than the yields from the DNeasy® method. Notably, the 500ml sample demonstrated the highest yield, although it was only 25% more than the 50ml and 200ml samples, suggesting a saturation of the PureYield™ binding column with processing volumes exceeding 200ml.

Furthermore, the purity assessment showed that all samples processed by the Promega Wizard® Enviro TNA Kit had consistently higher purity, with A₂₆₀/A₂₈₀ ratios of 1.7-1.8 and A₂₆₀/A₂₃₀ ratios of 1.0-1.2. In contrast, the Qiagen DNeasy® samples had A₂₆₀/A₂₈₀ ratios of 1.0-1.7 and A₂₆₀/A₂₃₀ ratios of 0.1-0.4, indicating a lower purity level.

For a more comprehensive analysis, all eDNA extracts were assayed using qPCR with the Promega GoTaq® Enviro RT-qPCR kit, adhering to the manufacturer's protocol. The technical reproducibility of the qPCR was evaluated with five technical replicates for all eDNA samples. To facilitate a direct comparison by qPCR, the concentrations of all samples were normalized by diluting aliquots of each sample to 2ng/µl and using 3µl of each sample, resulting in a total of 6ng input. Additionally, the Promega Inhibition Control assay was performed separately on the same sample dilutions to assess the inhibitor carryover for each extraction method.

Among the tested samples, six out of seven expected positive eDNA samples amplified with a C_T average of 36.3. The average ΔC_T between samples was approximately 1, with the highest ΔC_T of 2.9 observed between the highest Goby COI target amount (sample Q4) and the lowest Goby COI target amount (sample P1). However, the 500ml water Promega Wizard® Enviro TNA Kit 6ng input sample did not amplify, potentially due to the larger dilution factor (1:36.3) utilized to normalize this sample to 6ng.

Based on the known copy number of 18,000 copies of gBlock positive control DNA, it was estimated that the Round Goby COI gene copies per qPCR ranged from 10 to 80 copies per reaction using 6ng of total nucleic acid as input. This thorough analysis underpins the variances in eDNA extraction efficacy and purity between the two methods employed, while also providing critical insights into the technical reproducibility and potential inhibitor carryover in qPCR assays, which are pivotal for accurate eDNA analysis.

The comprehensive breakdown of the Promega Wizard® Enviro TNA Kit’s performance in eDNA analysis compared to that of the Qiagen DNeasy® Blood & Tissue Kit underscores the Wizard® Enviro TNA Kit’s utility in this application. The advantage of this kit lies primarily in the streamlined protocol, which allows for the processing of up to 20 water samples simultaneously using a vacuum manifold. This feature not only enhances throughput but also significantly reduces the total processing time to approximately one hour, with about 20–30 minutes of hands-on time, making the Promega Wizard® Enviro TNA Kit an efficient choice for large-scale studies.

One of the key strengths of the Promega Wizard® Enviro TNA Kit is the consistently high yield of eDNA across various sample volumes. With recovery rates typically ranging around 2–3 µg per sample for all input amounts, this consistency is crucial for studies requiring precise quantification and comparison across samples.

Moreover, the kit's minimal sample manipulation markedly reduces the risk of sample loss or contamination. This aspect is particularly critical in eDNA studies, where the integrity and purity of samples directly impact the accuracy and reliability of the findings. The superior sample purity achieved with this kit, as evidenced by favorable A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ ratios, further enhances its suitability for sensitive and accurate eDNA analysis.

While there is a slightly higher cost per extraction compared to the DNeasy® Blood & Tissue Kit, approximately $21 per sample, this is offset by the faster processing time and the inclusion of PCR inhibitor removal in the protocol, which can be a pivotal factor in successful eDNA amplification. However, it is important to note that the highest volume sample extracted (500ml) indicated possible saturation of the PureYield column, suggesting a need for careful consideration of sample volumes to optimize the recovery of target eDNA with the Wizard® Enviro TNA Kit.

The study evaluated two eDNA extraction kits: the DNeasy® Blood & Tissue Kit and the Promega Wizard® Enviro TNA Kit, assessing their efficiency, cost and suitability for eDNA analysis.

While the DNeasy® Blood & Tissue Kit offers cost-effectiveness and stability for transport and storage, its time-consuming nature and limited throughput pose significant drawbacks. The Promega Wizard® Enviro TNA Kit, on the other hand, features a streamlined protocol, consistent and higher eDNA recovery, and superior sample purity, despite its slightly higher cost and potential issues with column saturation in high-volume samples. The choice between these kits should be guided by the specific requirements of the study, considering factors like sample volume, desired throughput and budget constraints.

The Promega Wizard® Enviro TNA Kit presents itself as a highly effective and efficient solution for eDNA extraction and analysis. Its combination of speed, consistency and purity makes it an excellent choice for labs handling large sample sizes or requiring rapid throughput without compromising on the quality and integrity of the eDNA extracted.

Acknowledgements

Field samples for this study were kindly provided by Dr. Steven H. Pearson, Bureau of Invasive Species and Ecosystem Health, New York State Department of Environmental Conservation.