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Anastasija Zaiko, Ulla von Ammon, Jacqui Stuart, Kirsty F. Smith, Richard Yao, Melissa Welsh, Xavier Pochon, Holly A. Bowers (2022). Assessing the performance and efficiency of environmental DNA/RNA capture methodologies under controlled experimental conditions . Methods in Ecology and Evolution (accepted)

Abstract: Growing interest and affordability of environmental DNA and RNA (eDNA and eRNA) approaches for biodiversity assessments and monitoring of complex ecosystems have led to the emergence of manifold protocols for nucleic acids (NAs) isolation and processing. Although there is no consensus on a standardized workflow, the common practice for water samples is to concentrate NAs via filtration using varying pore size membranes. Using the smallest pore is assumed to be most efficient for NAs capture from a wide range of material (including sub-cellular particles), however a trade-off must occur between detection of a meaningful molecular signal and cost/time effort when processing samples using fine pore membranes. Comparative studies involving formal efficiency assessments are lacking, which restricts informed decision-making around an optimized sampling approach for applications such as biosurveillance (i.e., detection and monitoring of target taxa – nuisance organisms, endangered and indicator taxa or other species of economic or cultural importance). Here, we present an experimental study using an easily cultured microalgal species (Alexandrium pacificum) to test different filter membranes for capturing NAs in the context of cost/time effort and cell fractions encountered in nature (whole cells, partially lysed, and naked NAs). The results showed no statistically significant difference between membrane types for capturing target eDNA signal from intact and partially lysed cell treatments. In terms of time effort and volume processed, higher efficiency ratings were obtained with the larger pore size (5 µm) cellulose membranes. Positively charged nylon demonstrated enhanced capture of naked NAs, and especially eRNA signal, across treatments. Our findings support using coarse pore size filters for adequate capture of target NA signal (from both eDNA and eRNA) with less processing time. The framework presented here can provide a quick and robust feasibility check and comparative assessment of new and existing NA processing technologies, and allows sufficient control over multiple parameters, including physical-chemical water properties, temporal scales, and concentration and type of input material.

File descriptions: Raw ddPCR data collected during two experimental runs.

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