Comparison of capture and storage methods for aqueous macrobial eDNA using an optimized extraction protocol: advantage of enclosed filter

Abstract

Summary Aqueous environmental DNA (eDNA) is an emerging efficient non‐invasive tool for species inventory studies. To maximize performance of downstream quantitative PCR (qPCR) and next‐generation sequencing (NGS) applications, quality and quantity of the starting material is crucial, calling for optimized capture, storage and extraction techniques of eDNA. Previous comparative studies for eDNA capture/storage have tested precipitation and ‘open’ filters. However, practical ‘enclosed’ filters which reduce unnecessary handling have not been included. Here, we fill this gap by comparing a filter capsule (Sterivex‐GP polyethersulfone, pore size 0·22 μm, hereafter called SX) with commonly used methods. Our experimental set‐up, covering altogether 41 treatments combining capture by precipitation or filtration with different preservation techniques and storage times, sampled one single lake (and a fish‐free control pond). We selected documented capture methods that have successfully targeted a wide range of fauna. The eDNA was extracted using an optimized protocol modified from the DNeasy® Blood & Tissue kit (Qiagen). We measured total eDNA concentrations and Cq‐values (cycles used for DNA quantification by qPCR) to target specific mtDNA cytochrome b (cyt b) sequences in two local keystone fish species. SX yielded higher amounts of total eDNA along with lower Cq‐values than polycarbonate track‐etched filters (PCTE), glass fibre filters (GF) or ethanol precipitation (EP). SX also generated lower Cq‐values than cellulose nitrate filters (CN) for one of the target species. DNA integrity of SX samples did not decrease significantly after 2 weeks of storage in contrast to GF and PCTE. Adding preservative before storage improved SX results. In conclusion, we recommend SX filters (originally designed for filtering micro‐organisms) as an efficient capture method for sampling macrobial eDNA. Ethanol or Longmire's buffer preservation of SX immediately after filtration is recommended. Preserved SX capsules may be stored at room temperature for at least 2 weeks without significant degradation. Reduced handling and less exposure to outside stress compared with other filters may contribute to better eDNA results. SX capsules are easily transported and enable eDNA sampling in remote and harsh field conditions as samples can be filtered/preserved on site.

Acidity promotes degradation of multi-species environmental DNA in lotic mesocosms

“…The eDNA was extracted from the filters using a modified Qiagen Blood and Tissue DNeasy (Qiagen, Hilden, Germany) extraction method 48, 49 . In short, the Longmire’s solution was first removed by passing the Longmires through the filter membrane.…”

Environmental DNA metabarcoding: Transforming how we survey animal and plant communities

“…There are a number of recent studies that focus on the capture, preservation and extraction of eDNA, and the literature reviewed therein summarizes the important considerations and trade-offs that should be tested before a large-scale study is conducted (e.g., Deiner, Walser, M€ achler, & Altermatt, 2015;Renshaw, Olds, Jerde, McVeigh, & Lodge, 2015;Spens et al, 2017). Rather than reiterate those aspects here, we focus on primer choice and library preparation.…”

Mu-DNA: a modular universal DNA extraction method adaptable for a wide range of sample types

“…For example, Spens et al (2016) made use of DNeasy Blood and Tissue in their protocols for water filters, including Sterivex filters. We adapted and tested these protocols (data not shown) and found them to be easily changed to use a Mu-DNA: Tissue/Water protocol.…”

“…DNeasy PowerSoil, or aspects thereof, has been used for stomach, gut or faecal analysis of invertebrates (Knapp et al 2010, O'Rorke et al 2015, fish (Koinari et al 2013, Bolnick et al 2014, reptiles (Lau et al 2013, Colston et al 2015, birds (Vo andJedlicka 2014, Lewis et al 2016), mammals (Parfrey et al 2014, Ishaq andWright 2014) and, in particular, the Human Microbiome Project (Aagaard et al 2013). DNeasy Blood and Tissue has been used for studies of environmental DNA (eDNA) from water samples (Rees et al 2014, Spens et al 2016, Niemiller et al 2017. Although widely used, commercial kits are expensive and separate kits can be required for different sample types.…”