Optimised protocol for the extraction of fish DNA from freshwater sediments

Abstract

1. Monitoring fish is necessary for understanding population dynamics, tracking distribution patterns and evaluating conservation efforts. Molecular techniques targeting environmental DNA (eDNA) are now considered effective methods for detecting specific species or characterising fish communities. The analysis of DNA from lake‐surface sediments (sedDNA) can provide a time‐integrated sampling approach which reduces the variability sometimes observed in water samples. However, studies of sedDNA have had varying success in detecting fish. The present study aimed to determine the most effective extraction method for recovering fish DNA from lake‐surface sediments. 2. A literature review was undertaken to identify DNA extraction methods usedpreviously on aquatic sediments targeting aquatic and terrestrial animals. Fivemethods with various modifications were tested to establish their ability to des-orb extracellular DNA. Based on these results, two methods were selected andoptimised, and the recovery of fish sedDNA characterised using droplet digi-tal PCR assays targeting eel and perch (Anguilla australis, Anguilla dieffenbachii,Perca fluviatilis). A range of sediment masses (0.25–20 g) were assessed to estab-lish the optimal amount required to accurately assess fish sedDNA. 3. The DNA extraction methods found to be most effective at recovering ex-tracellular DNA spiked into small sediment masses (0.25 g) were the DNeasyPowerSoil DNA Isolation Kit (QIAGEN), and the ABPS protocol which involvedan initial alkaline buffer extraction followed by the PowerSoil extraction kit. Forlarger sediment masses (>0.25 g) the ABPS protocol or the DNeasy PowerMaxSoil Kit (QIAGEN) with an additional ethanol DNA concentration step (PMETprotocol) yielded the highest concentrations of target genes across a range oflake sediments. Larger sediment masses (≤20 g was tested) increased the likeli-hood of detection of fish in sedDNA. Optimisation of the ABPS protocol was re-quired (65°C incubation temperature, pooling of multiple PowerSoil extractions)to overcome technical challenges related to co-precipitation of organic content in lake-surface sediments. This optimised ABPS protocol was called the “LakesABPS protocol”. 4. We recommend the use of the Lakes ABPS protocol as it is cheaper than thePMET protocol. Additionally, after the first extraction step, the process can beautomated on a DNA extraction robot, allowing for higher sample throughput.A mass of 10 g is suggested, although higher detection is achieved with moresediment, a suite of challenges, such as co-precipitation of organic content, areencountered when the amount is increased.5. This study highlights the complexity of the extraction and detection of fishsedDNA from lake sediment, especially when it has a high organic content. Wehave optimised a DNA extraction method to overcome some of these complexi-ties that can be applied to a wide range of lake sediments.