Establishing the Signal above the Noise: Accounting for an Environmental Background in the Detection and Quantification of Salmonid Environmental DNA
Abstract
:1. Introduction
2. Materials and Methods
2.1. Experimental Study System and Design
2.2. eDNA Sample Collection and Filtration
2.3. Lab Sample Processing
2.4. Coho Salmon eDNA Assay (eONKI4) Design and Validation
2.5. Generation of Standard Curves
2.6. Statistical Analyses of Fish Density and Dilution Experiments
3. Results
3.1. qPCR Salmon eDNA Assay Design and Calibration to Copy Number
3.2. Determination of the LOB-B and LOD-B
3.3. Density Experiment Results
3.4. Dilution Experiment Results
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Sequence Type | Sequence (5′→3′) |
---|---|
Forward Primer | CCCTTTTGACCTCACAGAG |
Reverse Primer | GGTTCAGGGCAGTTAGTTCT |
Probe | FAM- TGCCCTCTTTTTCCTAGCCGAGTACG-ZEN/Iowa Black FQ |
Amplicon | CCCTTTTGACCTCACAGAGGGGGAGTCAGAATTAGTCTCCGGATCAATGTAGAA TATGCTGGAGGGCCCTTTGCCCTCTTTTTCCTAGCCGAGTACGCTAACATCCTTCTAA TAAATACACTCTCAGCCATCCTATTCCTAGGCGCATCCCACATCCCCGCTTTAC CAGAACTAACTGCCCTGAACC |
Species Name | Common Name | Species Abbreviation | Percent Detection by eONKI4 |
---|---|---|---|
Oncorhynchus kisutch | Coho Salmon | ONKI | 100% |
Oncorhynchus tschawytscha | Chinook Salmon | ONTS | 0% |
Oncorhynchus clarkii | Cutthroat Trout | ONCL | 0% |
Oncorhynchus gorbuscha | Pink Salmon | ONGO | 0% |
Oncorhynchus keta | Chum Salmon | ONKE | 0% |
Oncorhynchus mykiss | Rainbow Trout | ONMY | 0% |
Oncorhynchus nerka | Sockeye Salmon | ONNE | 0% |
Thymallus arcticus | Arctic Grayling | THAR | 0% |
Lithobates (Rana) catesbeiana | American Bullfrog | LICA | 0% |
Homo sapiens | Human | HOSA | 0% |
eONKI4 Limit of Detection | Number of Technical Replicates | |||||||
---|---|---|---|---|---|---|---|---|
3 | 8 | 16 | 24 | 32 | 48 | 64 | 96 | |
LOD | 1.15 | 0.43 | 0.21 | 0.14 | 0.11 | 0.07 | 0.05 | 0.04 |
LOD_lower | 0.85 | 0.32 | 0.16 | 0.11 | 0.08 | 0.05 | 0.04 | 0.03 |
LOD_upper | 1.77 | 0.66 | 0.33 | 0.22 | 0.17 | 0.11 | 0.08 | 0.06 |
Limits | Number of Technical Replicates | ||||||||
---|---|---|---|---|---|---|---|---|---|
3 | 8 | 16 | 24 | 32 | 40 | 48 | 64 | 96 | |
LOB-B 1 | 1 | 2 | 3 | 3 | 4 | 5 | 5 | 7 | 9 |
LOB-Bcn 2 | 0.46 | 0.33 | 0.24 | 0.15 | 0.15 | 0.15 | 0.13 | 0.13 | 0.11 |
LOD-B 2 | 2.29 | 1.05 | 0.62 | 0.40 | 0.35 | 0.32 | 0.27 | 0.25 | 0.20 |
LOD-B_lower 2 | 1.69 | 0.78 | 0.46 | 0.29 | 0.26 | 0.24 | 0.20 | 0.18 | 0.15 |
LOD-B_upper 2 | 3.55 | 1.62 | 0.96 | 0.61 | 0.54 | 0.50 | 0.41 | 0.39 | 0.30 |
Dilution Volume (kL) Treatment | Tank Number | Transformed Ct Estimate | Estimated Number of Copies per Reaction | Number of Samples per Tank | Number of Technical Replicates per Tank | Number of Detects for All Technical Replicates | Tank Detection | Treatment Detection |
---|---|---|---|---|---|---|---|---|
10 | 1 | 16.32 | 21.5 | 4 | 32 | 32 | Detect | Detect |
2 | 16.83 | 29.7 | 4 | 32 | 32 | Detect | ||
3 | 15.74 | 14.8 | 3 | 24 | 24 | Detect | ||
4 | 17.20 | 37.6 | 3 | 24 | 24 | Detect | ||
20 | 1 | 15.73 | 14.7 | 2 | 16 | 16 | Detect | Detect |
2 | 16.00 | 17.4 | 3 | 24 | 24 | Detect | ||
3 | 16.00 | 17.5 | 3 | 24 | 24 | Detect | ||
4 | 15.98 | 17.3 | 3 | 24 | 24 | Detect | ||
40 | 1 | 5.93 | 0.70 | 3 | 24 | 11 | Detect | Detect |
2 | 13.47 | 3.64 | 3 | 24 | 23 | Detect | ||
3 | 13.66 | 2.85 | 3 | 24 | 22 | Detect | ||
4 | 8.07 | 1.12 | 3 | 24 | 15 | Detect | ||
80 | 1 | 0.41 | 0.049 | 3 | 24 | 1 | No Detect | No Detect |
2 | 0.65 | 0.100 | 3 | 24 | 2 | No Detect | ||
3 | 1.99 | 0.209 | 3 | 24 | 4 | Detect* | ||
4 | 0.50 | 0.049 | 3 | 24 | 1 | No Detect | ||
160 | 1 | 0.00 | 0.000 | 3 | 24 | 0 | No Detect | No Detect |
2 | 0.34 | 0.049 | 3 | 24 | 1 | No Detect | ||
3 | 0.00 | 0.000 | 3 | 24 | 0 | No Detect | ||
4 | 1.49 | 0.153 | 3 | 24 | 3 | No Detect | ||
1000 | 1 | 0.00 | 0.000 | 4 | 32 | 0 | No Detect | No Detect |
2 | 0.46 | 0.049 | 3 | 24 | 1 | No Detect | ||
3 | 0.85 | 0.153 | 4 | 32 | 4 | No Detect | ||
4 | 0.44 | 0.074 | 4 | 32 | 2 | No Detect |
Tank Number | Sample Number | Transformed Ct Estimate | Estimated Copy Numbers per Reaction | Number of Technical Replicates per Sample | Number of Detects per Sample | Sample Detection | Tank Detection |
---|---|---|---|---|---|---|---|
1 | 1 | 6.64 | 0.79 | 8 | 4 | Detect* | Detect |
2 | 4.48 | 0.54 | 8 | 3 | Detect* | ||
3 | 6.66 | 0.79 | 8 | 4 | Detect* | ||
2 | 1 | 11.89 | 2.38 | 8 | 7 | Detect | Detect |
2 | 14.51 | 6.75 | 8 | 8 | Detect | ||
3 | 14.00 | 4.90 | 8 | 8 | Detect | ||
3 | 1 | 14.25 | 5.75 | 8 | 8 | Detect | Detect |
2 | 16.04 | 17.99 | 8 | 8 | Detect | ||
3 | 10.67 | 1.59 | 8 | 6 | Detect | ||
4 | 1 | 11.54 | 2.38 | 8 | 7 | Detect | Detect |
2 | 3.16 | 0.33 | 8 | 2 | Detect* | ||
3 | 9.51 | 1.59 | 8 | 6 | Detect |
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Hocking, M.D.; MacAdams, J.C.; Allison, M.J.; Bergman, L.C.; Sneiderman, R.; Koop, B.F.; Starzomski, B.M.; Lesperance, M.L.; Helbing, C.C. Establishing the Signal above the Noise: Accounting for an Environmental Background in the Detection and Quantification of Salmonid Environmental DNA. Fishes 2022, 7, 266. https://doi.org/10.3390/fishes7050266
Hocking MD, MacAdams JC, Allison MJ, Bergman LC, Sneiderman R, Koop BF, Starzomski BM, Lesperance ML, Helbing CC. Establishing the Signal above the Noise: Accounting for an Environmental Background in the Detection and Quantification of Salmonid Environmental DNA. Fishes. 2022; 7(5):266. https://doi.org/10.3390/fishes7050266
Chicago/Turabian StyleHocking, Morgan D., Jeffrey C. MacAdams, Michael J. Allison, Lauren C. Bergman, Robert Sneiderman, Ben F. Koop, Brian M. Starzomski, Mary L. Lesperance, and Caren C. Helbing. 2022. "Establishing the Signal above the Noise: Accounting for an Environmental Background in the Detection and Quantification of Salmonid Environmental DNA" Fishes 7, no. 5: 266. https://doi.org/10.3390/fishes7050266