Methods to maximise recovery of environmental DNA from water samples

Abstract

The environmental DNA (eDNA) method is a detection technique that is rapidly gaining credibility as a sensitive tool useful in the surveillance and monitoring of invasive and threatened species. Because eDNA analysis often deals with small quantities of short and degraded DNA fragments, methods that maximize eDNA recovery are required to increase detectability. In this study, we performed experiments at different stages of the eDNA analysis to show which combinations of methods give the best recovery rate for eDNA. Using Oriental weatherloach (Misgurnus anguillicaudatus) as a study species, we show that various combinations of DNA capture, preservation and extraction methods can significantly affect DNA yield. Filtration using cellulose nitrate filter paper preserved in ethanol or stored in a -20°C freezer and extracted with the Qiagen DNeasy kit outperformed other combinations in terms of cost and efficiency of DNA recovery. Our results support the recommendation to filter water samples within 24hours but if this is not possible, our results suggest that refrigeration may be a better option than freezing for short-term storage (i.e., 3–5 days). This information is useful in designing eDNA detection of low-density invasive or threatened species, where small variations in DNA recovery can signify the difference between detection success or failure.

Meta‐analysis supports further refinement of eDNA for monitoring aquatic species‐specific abundance in nature

“…New studies should take advantage of the increasing number of methodological papers being published on eDNA collection techniques, extraction techniques, and the statistical analysis of eDNA data (e.g., Lacoursière-Roussel, Rosabal, et al, 2016;Hinlo et al, 2017;Chambert, Takahara, Pilliod, Goldberg, & Doi, 2018) to optimize and standardize field, laboratory, and analytical practices. New studies should take advantage of the increasing number of methodological papers being published on eDNA collection techniques, extraction techniques, and the statistical analysis of eDNA data (e.g., Lacoursière-Roussel, Rosabal, et al, 2016;Hinlo et al, 2017;Chambert, Takahara, Pilliod, Goldberg, & Doi, 2018) to optimize and standardize field, laboratory, and analytical practices.…”

Comment: The Importance of Sound Methodology in Environmental DNA Sampling

“…Several recent studies have determined that filtration of larger water volumes (≥1 L) results in greater fish eDNA capture and detection than precipitation of eDNA from small water volumes (Piggott ; Hinlo et al. ; Spens et al. ).…”

Monitoring the eradication of the highly invasive topmouth gudgeon (Pseudorasbora parva) using a novel eDNA assay

“…In our study, the probability of detection by qPCR was c. 4-6 times higher with a large water volume (750 ml) compared to a smaller volume (15 ml). Yet, using the same laboratory and field protocols as for the 750 ml samples, would have likely the resulted in even lower eDNA concentrations (Hinlo et al, 2017;Rees et al, 2014). In addition, the 15 ml samples were stored and processed differently from the larger volume which could have been affected the results.…”

“…Filtration of 250 ml water volumes has been the recommended minimum volume of water to produce successful detection in a range of aquatic species(Dougherty et al, 2017;Goldberg et al, 2016;Hinlo, Gleeson, Lintermans, & Furlan, 2017;Olds et al, 2016). Water samples of 250 ml were collected in triplicate from 10 evenly distributed sampling points per pond.…”

“…DNA filtration and extraction took place in a designated eDNA area in a laboratory where no previous P. parva DNA or tissue had been handled. DNA pellets were extracted using DNeasy PowerLyzer PowerSoil kit (Qiagen®), which produces higher yields of DNA for this method of eDNA capture(Hinlo et al, 2017), following a standard protocol with a reduction in elution volume (from 60 to 50 µl, to increase DNA concentration). P. parva DNA from filter papers was extracted using Qiagen® DNeasy Blood and Tissue Kit (Qiagen), following the Qiagen Blood Spot extraction protocol with an adjustment to the elution volume (from 200 to 50 µl) to maximize DNA concentration.The 15 ml water samples were centrifuged at 6°C for 45 min at 5,000 g(Ficetola et al, 2008) and the supernatant was poured off to allow DNA extraction from the resulting pellet.…”