Water pre-filtration methods to improve environmental DNA detection by real-time PCR and metabarcoding

Abstract

Environmental DNA (eDNA) analysis is a novel approach for biomonitoring and has been mostly used in clear water. It is difficult to detect eDNA in turbid water as filter clogging occurs, and environmental samples contain various substances that inhibit the polymerase chain reaction (PCR) and affect the accuracy of eDNA analysis. Therefore, we applied a pre-filtration method to better detect the fish species (particularly pale chub, Opsariichthys platypus) present in a water body by measuring eDNA in environmental samples containing PCR inhibitors. Upon conducting 12S rRNA metabarcoding analysis (MiFish), we found that pre-filtration did not affect the number or identities of fish species detected in our samples, but pre-filtration through pore sizes resulted in significantly reduced variance among replicate samples. Additionally, PCR amplification was improved by the pre-filtration of environmental samples containing PCR inhibitors such as humic substances. Although this study may appear to be a conservative and ancillary experiment, pre-filtration is a simple technique that can not only improve the physical properties of water, such as turbidity, but also the quality of eDNA biomonitoring.

Fine‐tuning the performance of abundance estimation based on environmental DNA (eDNA) focusing on eDNA particle size and marker length

“…Additionally, the >3 μm pore size filter generally reduces filter clogging, increasing the filtration volume of the water samples considerably compared with smaller pore size filters (e.g., Kumar et al, 2021). Moreover, Takasaki et al (2021) recently compared the prefiltration performance on eDNA detection using various pore size filters (10-840 μm pore sizes). The result showed that 10 μm prefiltration effectively reduced PCR inhibitors (e.g., humic substances) in river water samples without decreasing the detectability of target eDNA.…”

Fine-tuning the performance of abundance estimation based on environmental DNA (eDNA) focusing on eDNA particle size and marker length

“…Additionally, the >3 μm pore size filter generally reduces filter clogging, increasing the filtration volume of the water samples considerably compared with smaller pore size filters (e.g., Kumar et al, 2021). Moreover, Takasaki et al (2021) recently compared the pre-filtration performance on eDNA detection using various pore size filters (10-840 μm pore sizes). The result showed that 10 μm pre-filtration effectively reduced PCR inhibitors (e.g., humic substances) in river water samples without decreasing the detectability of target eDNA.…”

Environmental DNA Methods for Ecological Monitoring and Biodiversity Assessment in Estuaries

“…This is especially important for eDNA sampling in shallow wetlands, where it can be difficult to sample from marshy shores and the sampling can stir up sediment. Wetland-specific methodological adaptations, for example additional water pre-filtration steps or an adjustment in filter pore size, might be needed based on conditions like turbidity (Goldberg et al 2018;Takasaki et al 2021) or optimization of DNA purification protocols to release DNA bound to suspended particles (Lever et al 2015). In addition, the unique environmental conditions in wetlands may alter eDNA dynamics and detections compared to other systems.…”