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Optimizing an eDNA protocol for estuarine environments: Balancing sensitivity, cost and time.

ABSTRACT: Environmental DNA (eDNA) analysis has gained traction as a precise and cost-effective method for species and waterways management. To date, publications on eDNA protocol optimization have focused primarily on DNA yield. Therefore, it has not been possible to evaluate the cost and speed of specific components of the eDNA protocol, such as water filtration and DNA extraction method when designing or choosing an eDNA protocol. At the same time, these two parameters are essential for the experimental design of a project. Here we evaluate and rank 27 different eDNA protocols in the context of Chinook salmon (Oncorhynchus tshawytscha) eDNA detection in an estuarine environment. We present a comprehensive evaluation of multiple eDNA protocol parameters, balancing time, cost and DNA yield. We collected samples composed of 500 mL estuarine water from Deverton Slough (38°11'16.7"N 121°58'34.5"W) and 500 mL from tank water containing 1.3 juvenile Chinook Salmon per liter. Then, we compared extraction methods, filter types, use of inhibitor removal kit for DNA yield, processing time, and protocol cost. Lastly, we used an MCMC algorithm together with machine learning to understand the DNA yield of each step of the protocol as well as the interactions between those steps. Glass fiber filtration was to be the most resilient to high turbidites, filtering the samples in 2.32 ± 0.08 min instead of 14.16 ± 1.86 min and 6.72 ± 1.99 min for nitrocellulose and paper filter N1, respectively. The filtration DNA yield percentages for paper filter N1, glass fiber, and nitrocellulose were 0.00045 ± 0.00013, 0.00107 ± 0.00013, 0.00172 ± 0.00013. The DNA extraction yield percentage for QIagen, dipstick, NaOH, magnetic beads, and direct dipstick ranged from 0.047 ± 0.0388 to 0.475 ± 0.0357. For estuarine waters, which are challenging for eDNA studies due to high turbidity, variable salinity, and the presence of PCR inhibitors, we found that a protocol combining glass filters, magnetic beads, and an extra step for PCR inhibitor removal, is the method that best balances time, cost, and yield. In addition, we provide a generalized decision tree for determining the optimal eDNA protocol for other studies in aquatic systems. Our findings should be applicable to most aquatic environments and provide a clear guide for determining which eDNA protocol should be used under different study constraints.

SUBMITTER: Sanches TM

PROVIDER: S-EPMC7241769 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Optimizing an eDNA protocol for estuarine environments: Balancing sensitivity, cost and time.

Sanches Thiago M TM Schreier Andrea D AD

PloS one 20200521 5

Environmental DNA (eDNA) analysis has gained traction as a precise and cost-effective method for species and waterways management. To date, publications on eDNA protocol optimization have focused primarily on DNA yield. Therefore, it has not been possible to evaluate the cost and speed of specific components of the eDNA protocol, such as water filtration and DNA extraction method when designing or choosing an eDNA protocol. At the same time, these two parameters are essential for the experimenta ...[more]

PMID: 32437479

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Project description:Background: Genetic and genomic selection programs require large numbers of phenotypes observed for animals in shared environments. Direct measurements of phenotypes like meat quality, methane emission, and disease susceptibility are difficult and expensive to measure at scale but are critically important to livestock production. Our work leans on our understanding of the “Central Dogma” of molecular genetics to leverage molecular intermediates as cheaply-measured proxies of organism-level phenotypes. The rapidly declining cost of next-generation sequencing presents opportunities for population-level molecular phenotyping. While the cost of whole transcriptome sequencing has declined recently, its required sequencing depth still makes it an expensive choice for wide-scale molecular phenotyping. We aim to optimize 3′ mRNA sequencing (3′ mRNA-Seq) approaches for collecting cost-effective proxy molecular phenotypes for cattle from easy-to-collect tissue samples (i.e., whole blood). We used matched 3′ mRNA-Seq samples for 15 Holstein male calves in a heat stress trail to identify the 1) best library preparation kit (Takara SMART-Seq v4 3′ DE and Lexogen QuantSeq) and 2) optimal sequencing depth (0.5 to 20 million reads/sample) to capture gene expression phenotypes most cost-effectively. Results: Takara SMART-Seq v4 3′ DE outperformed Lexogen QuantSeq libraries across all metrics: number of quality reads, expressed genes, informative genes, differentially expressed genes, and 3′ biased intragenic variants. Serial downsampling analyses identified that as few as 8.0 million reads per sample could effectively capture most of the between-sample variation in gene expression. However, progressively more reads did provide marginal increases in recall across metrics. These 3′ mRNA-Seq reads can also capture animal genotypes that could be used as the basis for downstream imputation. The 10 million read downsampled groups called an average of 104,386 SNPs and 20,131 INDELs, many of which segregate at moderate minor allele frequencies in the population. Conclusion: This work demonstrates that 3′ mRNA-Seq with Takara SMART-Seq v4 3′ DE can provide an incredibly cost-effective (<$25/sample) approach to quantifying molecular phenotypes (gene expression) while discovering sufficient variation for use in genotype imputation. Ongoing work is evaluating the accuracy of imputation and the ability of much larger datasets to predict individual animal phenotypes.

Dataset Information

Optimizing an eDNA protocol for estuarine environments: Balancing sensitivity, cost and time.

Publications

Optimizing an eDNA protocol for estuarine environments: Balancing sensitivity, cost and time.

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