Capturing marine microbiomes and environmental DNA: A field sampling guide

Abstract

The expanding interest in marine microbiome and eDNA sequence data has led to a demand for sample collection and preservation standard practices to enable comparative assessments of results across studies and facilitate meta-analyses. We support this effort by providing guidelines based on a review of published methods and field sampling experiences. The major components considered here are environmental and resource considerations, sample processing strategies, sample storage options, and eDNA extraction protocols. It is impossible to provide universal recommendations considering the wide range of eDNA applications; rather, we provide information to design fit-for-purpose protocols. To manage scope, the focus here is on sampling collection and preservation of prokaryotic and microeukaryotic eDNA. Even with a focused view, the practical utility of any approach depends on multiple factors, including habitat type, available resources, and experimental goals. We broadly recommend enacting rigorous decontamination protocols, pilot studies to guide the filtration volume needed to characterize the target(s) of interest and minimize PCR inhibitor collection, and prioritizing sample freezing over (only) the addition of preservation buffer. An annotated list of studies that test these parameters is included for more detailed investigation on specific steps. To illustrate an approach that demonstrates fit-for-purpose methodologies, we provide a protocol for eDNA sampling aboard an oceanographic vessel. These guidelines can aid the decision-making process for scientists interested in sampling and sequencing marine microbiomes and/or eDNA.

What is environmental DNA?

“…These larger pore sized membranes yielded between 247% and 277% greater richness than a standard 0.45 μm membrane. This finding may seem counter‐intuitive, considering that bacteria are generally around 0.2–2.0 μm in diameter and 2–8 μm in length (Tortora et al., 2007) and the widespread use of smaller pore filters to characterize marine microbial communities (typically, 0.22 or 0.45 μm membranes) (Cottrell & Kirchman, 2004; Mestre et al., 2017; Needham et al., 2013; Patin & Goodwin, 2023). Yet, it accords with previous comparative work (Byappanahalli et al., 2021; Mestre et al., 2017), and likely reflects that bacteria rarely exist as free‐living entities; they often aggregate and form communities within biofilms (Berlanga & Guerrero, 2016; Davey & O'toole, 2000; Mestre et al., 2017).…”

“…Morphologically diverse taxa, such as algae, displayed relatively even coverage across the fractions, which may be attributed to different life phases of algae (e.g., chain‐forming algae vs. unicellular algae) and the high presence of cell fragments observed on the membranes. A broad range of optimal filter pore sizes have been prescribed previously for the collection of phytoplankton (0.2, 0.45, 0.6, 0.8, 1, 2, 3, or 5 μm; Patin & Goodwin, 2023; Vaulot et al., 2008). Our results indicate that the taxonomic and morphological diversity seen within this taxa group make it difficult to identify an optimal filter pore size to suit all needs for eDNA of phytoplankton.…”

“…These larger pore sized membranes yielded between 247% and 277% greater richness than a standard 0.45 μm membrane. This finding may seem counter-intuitive, considering that bacteria are generally around 0.2-2.0 μm in diameter and 2-8 μm in length (Tortora et al, 2007) and the widespread use of smaller pore filters to characterize marine microbial communities (typically, 0.22 or 0.45 μm membranes) (Cottrell & Kirchman, 2004;Mestre et al, 2017;Needham et al, 2013;Patin & Goodwin, 2023).…”

Advancing ecological assessment of the Arabian Gulf through eDNA metabarcoding: opportunities, prospects, and challenges

“…Elevated levels of PCR inhibitors such as humic acid and tannic acid in sediment samples collected from wetlands, coral reefs, and fish landing sites in the Gulf can affect PCR analyses. Therefore, it is necessary to either dilute the eDNA or implement additional purification steps to eliminate these inhibitors (Patin and Goodwin, 2023). As eDNA samples are highly heterogeneous, it is challenging to achieve a complete primer-target match during amplification, which could lead to biased PCR results (Stadhouders et al, 2010).…”

Environmental DNA: Enhancing Species Detection and Study Comparability with Synthetic DNA Tracers

“…A variety of studies have addressed sample collection approaches in both aquatic and terrestrial environments, focusing on strategies for filtration 84 ; sample preservation [85][86][87] ; and other aspects of physical collection and handling. These considerations should apply to any study of eDNA, but are particularly important for metabarcoding studies and those that attempt to amplify multiple DNA targets within a sample.…”