Environmental
DNA protocol
development
guide forbiomonitoring© University of Canberra 2022
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Inquiries about the licence and any use of this document should be emailed to EcoDNA@Canberra.edu.au.

Citation
De Brauwer M, Chariton A, Clarke LJ, Cooper MK, DiBattista J, Furlan E, Giblot-Ducray D, Gleeson D, Harford A,
Herbert S, MacDonald AJ, Miller A, Montgomery K, Mooney T, Noble LM, Rourke M, Sherman CDH, Stat M,
Suter L, West KM, White N, Villacorta-Rath C, Zaiko A & Trujillo-Gonzalez A (2022). Environmental DNA protocol
development guide for biomonitoring. National eDNA Reference Centre, Canberra.
ISBN 978-1-74088-532-4
Disclaimer
The Australian Government, acting through the Department of Agriculture, Water and the Environment,
has exercised due care and skill in preparing and compiling the information and data in this publication.
Notwithstanding, the Department of Agriculture, Water and the Environment, its employees and advisers
disclaim all liability, including liability for negligence and for any loss, damage, injury, expense or cost
incurred by any person as a result of accessing, using or relying on any of the information or data in thispublication to the maximum extent permitted by law.
Acknowledgements
This publication was developed as a collaborative project, funded by the Australian Government
Department of Agriculture, Fisheries and Forestry, and led by Alejandro Trujillo-Gonzalez (ATG) of the
University of Canberra and Maarten De Brauwer (MDB) of CSIRO. Anastasija Zaiko​‘s contribution was
supported by the New Zealand Ministry of Business, Innovation and Employment funding (CAWX1904 — A
toolbox to underpin and enable tomorrow’s marine biosecurity system). We would like to acknowledge
and thank all co-authors who helped develop and write the guidelines, as well as the 53 experts fromacross Australia and New Zealand who provided feedback during various stages of the process. The
authors thank the four external expert reviewers of the guidelines for their valuable feedback. ATG
and MDB would also like to thank Biotext for their services and Paula Doyle for her invaluable role asknowledge broker.
Editing and design: Biotext Pty Ltd

Contents
Glossary 

4Introduction 

6Principles for conducting an eDNA project 

9Principle 1: Ensure processes are fit for purpose 

9Principle 2: Test and validate processes 

13

Principle 3: Ensure good chain of custody and documentation 

14

Principle 4: Understand the limitations of results 

14Principle 5: Ensure good communication 

15

Principle 6: Recognise First Nations peoples’ ownership andstewardship 

15Environmental DNA/RNA test protocols 

161

Experimental design 172Collect samples 

23

3

Preserve samples for processing 

28

4

Extract and isolate DNA/RNA 

30

5Ensure quality control and purification of extracts 30

6Analyse extracts 

33

7Interpret and communicate results 40

Summary of key steps for environmental DNA/RNA test protocols 

41Resources 

43

References 45Glossary
Assay: The laboratory workflow from DNA extraction to sequence outputs.
Often refers to the target gene and taxonomic group (e.g. 16S_Fish,
18S universal, COI).
Endogenous control: Endogenous controls refer to either exogenous DNA(i.e. DNA that is spiked in) or endogenous DNA (i.e. DNA that is naturally
occurring) that can be targeted in environmental samples as positive
controls to monitor method success. Exogenous DNA templates can begenerated from custom-synthesised DNA fragments, DNA extract, plasmids,
and be added to samples during any stage of the eDNA workflow after
sample collection. Endogenous controls use the fact that DNA is ubiquitousin the environment, such that every environmental sample will contain DNA
from multiple sources. Within this context, a generic primer assay can be
designed to amplify abundant, non-target DNA that will be simultaneously
sampled, captured, extracted and amplified with the target species’ DNA(Furlan & Gleeson 2016).
Environmental DNA / RNA (eDNA / eRNA): DNA or RNA directly extracted
from environmental samples (soil, sediment, water, etc.) without any
knowledge of the original organism. DNA carries genetic material, while RNAtransfers information within cells to produce specific proteins and is only
shed by physiologically active (living) organisms.
High-throughput sequencing (HTS): A technique able to determine thenucleotide composition of millions of nucleic acid sequences. Different
platforms of sequencing are available including sequencing by synthesis (e.g.
Illumina), single molecule real time (e.g. PacBio), and nanopore (e.g. Oxford
Nanopore Technologies).
Inhibitory substances: Substances in a sample or extract that reduce assay
sensitivity and increase the risk of false negative results in a PCR test.
Library (also Reference library, Reference database): Database with DNA
sequences of specific species.

Environmental DNA protocol development guide for biomonitoring

4

Limit of detection (LOD): The lowest concentration of target DNA that canbe detected with a defined level of confidence (usually a 95% detection rate).
Limit of quantification (LOQ): The lowest amount of DNA in a sample
that can be quantitatively determined with a stated precision, under statedexperimental conditions.
Metabarcoding: Simultaneous taxonomic identification of Operational
Taxonomic Units (OTUs) or Amplicon Specific Variances (ASVs) in eDNA
samples with millions of sequences, generated by PCR amplification using
one of the HTS techniques.
Monitoring: The systematic collection of data over time to detect changes
in a system (Gerber et al 2005). Data can include information on a range offactors such as environmental, ecological, biological and social.
Polymerase chain reaction (PCR): A molecular technique that allows
the exponential amplification of a target fragment/region of DNA froma mixture of DNA fragments. The desired fragment is selected from theother fragments in the mixture by specific primers (small single-strand
oligonucleotides) complementary to the desired sequence.
Primer: Short DNA fragments used in PCR amplification that bind adjacentto the target region or gene.
Quantitative PCR (qPCR): A variant of PCR. The main difference is that qPCR
is able to quantify how many fragments of DNA are amplified during eachstep in the reaction, leading to quantitative data.
Sequencing: Determining the order of nucleotides in DNA or RNA; this canbe done using a variety of methods.

Environmental DNA protocol development guide for biomonitoring5Introduction
The use of environmental DNA (eDNA) and RNA (eRNA)methods is a rapidly advancing field that provides fast,
cost-effective, non-invasive methods to identify the presenceof target species. These methods can be used, for example, toscreen for pest species as part of biosecurity measures andrisk management, to screen for threatened species as partof development requirements, or for biodiversity monitoring.
In an operational context, high-quality standard assurance
requirements are needed to ensure that assays and protocolsdeliver reliable results across multiple applications and purposes.

What is the aim of these guidelines?
The Environmental DNA protocol development guide for biomonitoring (EPguide for biomonitoring) provides harmonised quality control and minimum
standard operating procedures. This document is complemented by theEnvironmental DNA test validation guidelines, which has been published inparallel with the EP guide for biomonitoring. The EP guide for biomonitoring
provides information to create standard operating procedures for eDNA/
eRNA projects, whereas the eDNA test validation guidelines focus on advice
for the development and use of eDNA and eRNA assays.
This document is a comprehensive guide for the development and use
of eDNA/eRNA test protocols, as recommended and curated by experts,
stakeholders and end users in Australia and New Zealand. The EP guide forbiomonitoring covers protocols for both single species (qPCR) and multispecies (metabarcoding) projects. Differences between approaches arehighlighted in relevant sections throughout the guidelines. The guide isdesigned to support a consistent and best-practice approach to eDNA/eRNA

Environmental DNA protocol development guide for biomonitoring6

testing to help detect species of interest. This approach ensures thatsurveillance and resource managers are provided with robust scientificevidence to support decision making.
Environmental RNA-based methods differ primarily from eDNA in themolecules they target. The different function of RNA in living organisms
means that its detection is more likely to reflect metabolically active
organisms. Current eRNA applications exploit its rapid degradation toquantify temporally recent or metabolically active communities (Pokon et al.
2017, Yates et al. 2021), providing important information to assess the active
presence of species in an environment.
Note on the use of the terms eDNA and eRNA: Protocols for the use of eDNAand eRNA-based methods have considerable overlap; we therefore use theterm ‘eDNA’ throughout the text when protocols are applicable to both eDNAand eRNA methods. Specific eRNA protocols are discussed separately whendifferent protocols are required.

Who are they for?
The EP guide for biomonitoring provides clear best-practice benchmarks tohelp researchers develop eDNA-based protocols encompassing complete
workflows for reliable eDNA analysis. It also provides information for
end users requesting eDNA/eRNA projects.
The potential applications for eDNA are varied and eDNA-based methods
have been applied in a wide range of fields, including biosecurity
surveillance, biodiversity monitoring, endangered species research, risk
management, and emergency response. The use of these guidelines for
protocol development is strongly recommended, but not mandatory.

For researchers
The EP guide for biomonitoring details key steps in creating standard
operating procedures for eDNA-based applications. This document also
provides general principles and considerations to guide project development,
and information on other important project areas such as communicationand ethics.

For end users
The EP guide for biomonitoring provides quality assurance for any contracted
eDNA work. The guide tells end users what services and standards can be
Environmental DNA protocol development guide for biomonitoring

7

expected and may also be used to inform staff collecting samples or informthe development of other areas of the project.

How have they been developed?
The EP guide for biomonitoring and the Environmental DNA test validation
guidelines were developed in a collaborative process with input from eDNA
experts and end users from across Australia and New Zealand. Initial draft
frameworks were developed and led by members of the Southern eDNA
Society Standards and Best Practices Committee, after which multiple
consultation rounds with experts, end users and stakeholders from private
entities and public agencies were held to adapt the frameworks to meet
Australian and New Zealand needs. Three consultation periods with eDNA
experts, private stakeholders, government officials and end users were heldin 2021–22 to ensure the guidelines were fit for purpose and met the highest
quality standards in the field.

Updates
We recognise that eDNA methods and resources are still rapidly evolving. It is
anticipated that the EP guide for biomonitoring will be updated and expanded
over time, with a review and update as required.
The guidelines will be reviewed and updated by the Australian National
eDNA Reference Centre, with input from leading experts in the field ofenvironmental DNA.

Environmental DNA protocol development guide for biomonitoring

8

Principles for conducting an
eDNA project
The EP guide for biomonitoring provides best-practice guidance, while
recognising that ‘best practice’ is broad and depends on:• purpose of the test (e.g. the detection of high-impact pests may require amore sensitive detection threshold than is used for low-impact species)• environmental constraints (e.g. sample type - water, soil or scats; integrity)• target constraints (e.g. mobile or sedentary target, concentration)• project constraints (e.g. budget, sample shipping, turnaround time
requested)• current knowledge gaps (e.g. in taxonomic groups or DNA reference
sample data).
Best practice will therefore involve various considerations, taking 6 generalprinciples into account:
1 Ensure processes are fit for purpose
2 Test and validate processes
3 Ensure good chain of custody and documentation
4 Understand the limitations of results
5 Ensure good communication
6 Recognise First Nations peoples’ ownership and stewardship.

Principle 1: Ensure processes are fit for
purposeThe fundamental steps of conducting an eDNA project do not differ
substantially from other research or monitoring projects. As with othermethods, projects need a clear goal and experimental design tied to theproject goal, samples must be collected and processed, and results must be
interpreted and communicated to a target audience or end users (Figure 1).

Environmental DNA protocol development guide for biomonitoring

9

Projectgoal

Designexperiment

CollectsamplesLaboratory
analysesData
analyses

Interpret
results

Do the results meet the project goal?

Figure 1 Steps of an eDNA project, and how they must consider the project
goal if they are to be fit for purpose
It is important that processes at each of these steps are fit for purpose.
Considerations include:• project goal and experimental design (see Experimental design) – match all
aspects of the experimental design to the purpose of the project• sample collection and preservation (see Collect samples and Preserve
samples for processing) – design and provide appropriate guidelines andworkflows to suit the project purpose and the constraints of the site; train
any non-experts involved in collecting samples• recording all sample metadata, from collection through to projectcompletion• laboratory analyses (see Extract and isolate DNA/RNA and Ensure quality
control and purification of extracts) – laboratory processes and methods
must be optimised for eDNA and carried out using strict standard
operating procedures• data analyses (see Analyse extracts) – data analyses must be able toclearly identify positive, negative, indeterminate and unreliable results• documenting all of the above processes (including sample storage
and preservation, and laboratory protocols, optimisations andtroubleshooting) for reporting to the end user
• result interpretation (see Interpret and communicate results) –interpretation of resuts must take into account any limitations and beassessed and compared with the project goals; conclusions should
be communicated as clearly as possible, with reference to the project
goal; any caveats around the results or interpretation should be clearlycommunicated in the report• follow-up and confirmation of positive results.
Suggested considerations for each stage of the project workflow areprovided in Table 1.

Environmental DNA protocol development guide for biomonitoring

10

Table 1 Guiding questions for consideration in the design andimplementation phases of an environmental DNA project
Workflow stage Guiding questions
Study design

In the field•Is the work basic science or applied (e.g. environmental
biomonitoring)?

•

What is your study goal (e.g. presence or absence; diversity;
quantification)?

•

What tools and methods are available and what are yet to
be developed? (e.g. sampling methods, assays, analysis
pipelines)?

•

What is the best analytical approach to address
the research goal (e.g. DNA-based or RNA-based,
metabarcoding vs qPCR)?

•

Which taxa will you target?

•

What characteristics of the target organism/communities
may affect the sampling method (e.g. life form, size,
expected abundance/occupancy, behaviour)?

•What characteristics of the ecosystem may affect the
sampling method (e.g. terrestrial/aquatic; lentic/lotic/
marine; how variable is the environment)?

•

Is the scale of inference for your sample type appropriate
to your questions?

•

Can you compare complementary data types(e.g. traditional vs eDNA)?

•

Does your sampling/replication scheme provide good
statistical power?

•

Who are the end users and what will they do in response to
eDNA results? Are they involved in project design?

•

What type of sample (water, soil, air) is needed?

•

What metadata will you collect?

•

How many replicates will you collect?

•

Does your sampling protocol minimise or control for– contamination (e.g. positive and negative controls)?
– any known biases (e.g. inhibitors, sample volume)?

•

How will you track samples (e.g. barcodes)?

•What is the most appropriate sample collection method forthe established goal/ecosystem/target organisms basedon the characteristics of the target species/community andecosystem?

•

How will samples be preserved and transported?
continues

Environmental DNA protocol development guide for biomonitoring

11

Table 1

continued

Workflow stage Guiding questions
In the laboratory•

What extraction method (physical vs chemical)?

– sample•

How much sample volume or weight?

handling phase•

How many samples?

•

How many controls and of what sort?

•

Is species-specific detection or metabarcoding more
appropriate?

•

Is a species-specific assay available? Does it need validation?

•

Do you need to generate reference sequence data?

•

Are any technical replicates needed?

•

For eRNA analyses, will you use co-extraction or separate
extraction?

•

For qPCR/ddCPR, choose the assay for the target species(e.g. is it validated for your region, checked for crossreactivity with non-target taxa)?

•

What storage is needed?
– How long will samples be stored before processing?
– What are the most appropriate conditions for sample
type?
– What preservation should be used?
– How will products (DNA/RNA extracts, PCR amplicons)
be stored and for how long?

•

How can multiple freeze–thaw cycles for products beminimised?

•

Is contamination control implemented at different steps(extraction blanks, PCR blanks, etc.) and included in theanalyses?

•

What sequencing platform/qPCR instrument (96/384 well)
will you use?

DNA processing•

What locus and primers will you use?

phase

•

What read length will you use?

•

Do you need paired-end sequencing?

•

Have you included appropriate quality assurances?
(e.g. internal control, mock community, qPCR, bioanalyser
tracer)•

Does your sampling protocol minimise or control for

In the laboratory–– contamination (e.g. positive and negative controls)?
– any known biases (e.g. primer bias, coverage, taxonomic
resolution)?
continues

Environmental DNA protocol development guide for biomonitoring

12

Table 1

continued

Workflow stage Guiding questions
At the keyboard•

How complete is the reference database?

•

How complete is the taxonomic knowledge for the group?
Are there risks of taxonomic uncertainty that could affect
how results are interpreted?

•

Do you have adequate sequencing coverage acrosssamples?

•

Are you using appropriate choices for software tools andparameters?

•

Are your conclusions upheld when using alternative
parameters and workflows?

•

Are you including appropriate quality filtering of your data?

•

Is the specificity of the test confirmed?

•

What is the LOD/LOQ of the test?

•

Are all the internal controls / QA controls valid?

•

Is sequencing data of adequate quality (sequence depth
per sample and error rates adequate, and contamination
negligible)?

•

Which sequencing database will you use? How complete
and up to date is it?

•

How much ambiguity is in the taxonomic assignment(i.e. can your data provide required taxonomic resolution
with high confidence?)

Source: adapted from Deiner et al. 2017.

Principle 2: Test and validate processes
Processes should be tested and validated to ensure robust results. Consider
doing a pilot project, which may help to determine sample sizes andprotocols.
Validation requirements will depend on the study purpose. For more
information and detailed guidelines, see the Environmental DNA test validation
guidelines, published in parallel with this document. Other scales andguidelines include:• eDNA Validation Scale: edna-validation.com• Thalinger B, Deiner K, Harper LR, Rees HC, Blackman RC, Sint D,
Traugott M, Goldberg CS, Bruce K (2021). A validation scale to determine
the readiness of environmental DNA assays for routine species
monitoring. Environmental DNA 3(4):823–836, doi:10.1002/edn3.189.
Environmental DNA protocol development guide for biomonitoring13• Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M,
Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT(2009). The MIQE guidelines: minimum information for publication ofquantitative real-time PCR experiments. Clinical Chemistry 55(4):611–22,
doi:10.1373/clinchem.2008.112797.
Processes or results can also be validated by crosschecking with otherreputable laboratories.

Principle 3: Ensure good chain of custody anddocumentation
Good chain of custody and documentation (see Collect samples) ensuresresults can be trusted. Documentation of processes can help to ensure
processes are robust and consistent and are followed correctly. Reliable
tracking systems ensure the reliability of metadata collected in the field.

Principle 4: Understand the limitations ofresults
The limitations of eDNA results will be project-specific, and depend on theenvironment, target species and assays used. Limitations of results should
be considered during experimental design and – most importantly – analysis
and interpretation phases.
Limitations of results will be closely linked to the purpose of the project andthus to the stringency required (see Purpose and stringency). Projects withhigh stringency requirements (e.g. legal evidence), must take into accountand communicate the limitations of results (see Interpret and communicate
results).
In some cases, eDNA testing can be used for large-scale surveillance, toenable triage and better use of resources for targeted traditional surveys.
Data obtained from eDNA methods might not necessarily confirm thepresence or absence of a species, but positive eDNA tests are appropriate
criteria to identify the potential presence of target species and trigger
confirmatory efforts.
When eDNA methods are used to study communities using metabarcoding,limitations such as incomplete reference databases or unresolved taxonomy
issues must be taken into account when interpreting results.

Environmental DNA protocol development guide for biomonitoring

14

Principle 5: Ensure good communication
Clear communication, especially with service providers of the project and withproject members without genetics expertise, is an essential part of project
protocols, and will support best-practice processes. Clear communication isparticularly important with:• End users (see Interpret and communicate results). Clear communication
throughout the project will help to ensure expectations are managed and theresults meet user needs. It is important to emphasise limitations and explain
uncertainties in results to ensure data are not misinterpreted.
• Collection teams (see Collect samples). Collection teams may not be trained as
molecular scientists and will need clear, non-technical guidance about how tocollect, preserve, store, transport, and document samples. In-person training
may improve sample collection standards.

Principle 6: Recognise First Nations peoples’
ownership and stewardshipCollection of environmental samples in Australia and New Zealand requires
recognition of the relationship that First Nations peoples have with nature.
Following the release of the global biodiversity framework from the United Nations
Convention on Biological Diversity in 2021, and acknowledging the rights of First
Nations peoples with respect to traditional knowledge associated with genetic
resources, these guidelines highlight the following:• There are international and national efforts to recognise the rights ofIndigenous peoples with respect to the traditional knowledge associatedwith genetic resources.
• These guidelines recognise the need for fair and equitable sharing of thebenefits accrued from gathering genetic resources and their associated digital
information. Sharing strategies should be, among other things, efficient,feasible and practical. They should aim to be effective and not hinderresearch and innovation, while being mutually supportive of other accessand benefit-sharing instruments.
• There are a wide range of views about the modalities and methodologies ofa potential solution for the fair and equitable sharing of benefits arising fromdigital sequence information from genetic resources.
With these points in mind, and aligned with recommendations made by theConvention on Biological Diversity, these guidelines recommend that project
designers discuss projects with appropriate First Nations communities and provide
transparency on project requirements and outcomes. Discussions with Traditional
Custodians about the collection and use of environmental samples must be flexible- listen to, adapt and incorporate recommendations from the Traditional Custodiansof study sites. It is also recommended that project outcomes are shared with First
Nations communities and, when possible, the Traditional Custodians are recognised
and acknowledged in online repositories that store genetic data.
Environmental DNA protocol development guide for biomonitoring

15

Environmental DNA/RNA test
protocols
Environmental DNA/RNA test protocols outline standard operatingprocedures for field officers and users to collect, extract and analyse
environmental samples for eDNA/eRNA. These protocols encompass theentire eDNA workflow and offer users standard guidelines for each stage,
with appropriate controls and measures for independence. The protocols
can be used as templates to develop standard operating procedures (SOPs)
for specific projects and purposes. They align with and meet the minimum
requirements of the:• Development of National Diagnostic Protocols – Procedures for Authors
Reference Standard (SPHD RS No. 2), approved by the Subcommittee on
Plant Health Diagnostics (SPHD, 2019)• Australian and New Zealand standard diagnostic procedures – Guide for
Authors (ANZSDP, 2021).
Each of the following steps includes important considerations for quality
assurance and reliable implementation and analysis of eDNA/eRNA testing:
1 Experimental design
2 Collect samples
3 Preserve samples for processing
4 Extract and isolate DNA/RNA
5 Ensure quality control and purification of extracts
6 Analyse extracts
7 Interpret and communicate results

Environmental DNA protocol development guide for biomonitoring

16

1

Experimental design

Careful, detailed experimental design is an essential investment to ensure
projects are effective and meet end-user needs. eDNA analyses provide
powerful molecular tools with a wide range of biomonitoring applications,but a well-thought-out experimental design is essential to guarantee
high-quality outcomes. Poorly designed experiments or surveys are more
likely than not to compromise results, regardless of the quality of assays,laboratory protocols and bioinformatics pipelines.
• Experimental protocols should be designed in a way that complements
the principles for conducting projects (see Principles for conducting an
eDNA project).
Standard operating procedures should implement suitable sampling regimes
determined during initial experimental design (Table 2). This initial stepshould assess:• Purpose of test (see Purpose and stringency). Is the test intended to
be used for a purpose that requires highest-level standards (e.g. legal
purposes), survey biodiversity, or for routine biodiversity monitoring?
What level of detection is meant to be achieved? What level of uncertainty
is acceptable?
• Responsibility. Who is responsible for ensuring experimental design is fit
for purpose?
• Spatial extent. How many samples should be collected from how manysites to achieve suitable representation of the environment and presumed
distribution of the target species?
• Temporal extent. Are you collecting samples in a single session, or
multiple sessions over time? Will the time of sampling affect the presenceor abundance of target species (e.g. day, night, season)?
• Collection method. What collection method should be used to collectsamples within the target environment?
• Replication. What volume of sample and how many replicates should becollected to improve detection probability?
• Preservation. What method is the most appropriate to preserve samples
until eDNA/eRNA extracts can be obtained?
• Controls. Which controls will be implemented during different stages of
the workflow?
• Confirmation. What method can be used to confirm positive results?

Environmental DNA protocol development guide for biomonitoring17• For long-term programs, conducting a pilot study to inform proper study
design, cost effectiveness, detection probabilities, and spatial distributionof eDNA of target species can be helpful. If relative abundance is includedin the purpose, pilot studies and calibration with existing methods isessential.
The experimental design of a project should be done in consultation
with the end users of the data to ensure the project workflow is fit for
purpose. Before the project begins, researchers should communicate
clearly and honestly about how and if eDNA can meet the project goals.
This includes clearly explaining what eDNA testing is capable of andinforming end users about the constraints and limitations of eDNA methods.
Managing expectations around constraints such as abundance data, poorly
amplifying taxa, and so on, is essential to ensure the eDNA project will meet
end-user needs.
Table 2 Factors to consider when designing an eDNA/eRNA study
FactorConsiderations

ApproachPurposeEnd-user goal

Projects with different

Consideration of otherpotential sources of DNA(e.g. could the DNA originate
from elsewhere?)

purposes (e.g. routine
biomonitoring vs biosecurity)
might require different levels ofstringency and controls

Ethics andPermits might be required

Consult with appropriate

permits

to collect samples (e.g. inauthorities

national parks)
Outcomes of surveys mightConsult relevant First Nations

have upstream implications

representatives

for First Nations peoples
BudgetSpatial andBudget might not be sufficientRealistic estimates to meet user

to achieve intended purpose

needs

Single site vs large scale,

Design for maximum

temporal extent single time point vs long-term

reproducibility andsampling

transferability

Consider using statistical

Consider minimum number of(probabilistic) models andsites to reach adequate spatial

simulations to test your study

coveragedesign in silico
continues

Environmental DNA protocol development guide for biomonitoring

18

Table 2

continued

FactorConsiderations

Approach

Environmental
factors

Temperature, UV exposure,water movement, turbidity,
chlorophyll a, biochemical
oxygen demand, dissolved
oxygen, pH

Different conditions require
specific methods and materials

MediumWater, sediment, soil, grain,scat, stomach content, air

Different media require specificfield and laboratory protocols

Controls

Controls should be
implemented at different
workflow stages

Different purposes andworkflows might requiredifferent controls

Targetrestrictions

Biology and behaviour of thetarget, likely levels of DNA
in the environment, risk ofcryptic species

Consider potential issues
that will affect the samplingstrategy, analysis, andinterpretation of the data

Differential amplification
between target taxa
Fieldwork
restrictions

Facilities, distance toprocessing laboratories,
contamination sources andcontrols, extraction andpreservation options

Consider all needs andpotential issues

Sample tracking options
Laboratory
processing

Assay

Choice of sample collection
method (e.g. filtration,
sediment core)

Consider benefits anddrawbacks of different sample
collection methods at eachstage of the project

Choice of preservation
methodConsider logistics and effects
of preservation method on
sample quality and extraction
protocolChoice of DNA extraction
methodConsider logistics and effects ofextraction method

Assays fit for purpose

Validated assay specific totarget species
Metabarcoding primers
validated to detect taxa ofinterest
continues

Environmental DNA protocol development guide for biomonitoring

19

Table 2

continued

FactorConsiderations

Approach

Replication

Need adequate replication

Higher replication needed forfor different environments orcertain purposes requiring

purposehigher levels of stringency(e.g. surveys with potential
legal implications)
More biodiverse regions needhigher sample replication

Staff training

Collection teams might not be

Ensure all relevant staff

needs

experts

members are adequately

Chain of custody, ensure eachtrainedlink in chain knows protocols

Purpose and stringency
Defining the intended purpose of a study should be the first step indesigning any eDNA project. A thorough understanding of the purpose of
eDNA testing is essential; it will form the framework around which the restof the experimental design is built. The purpose of the study will help to
guide decisions around the level of required stringency and what level ofrisk can be tolerated when taking management decision based on eDNA
data (Table 3). Projects with legal or high-cost ramifications –such as forbiosecurity regulation purposes – are likely to need higher standards andstricter protocols than, for example, citizen science projects that aim tomonitor biodiversity.
Table 3 A matrix for assessing the quality or stringency of an eDNA project
Quality or stringency
Aspect ofproject

High (desirable)

Medium (minimum
requirements)Low(insufficient)

Experimental

Power analysis

Robust design and

Ad hoc sampling

designdone and sufficient

some replication

without

sample size to answer

replicationquestion
Assay design

Proven assay with

Validated assay

known specificity andAssay has notbeen tested

sensitivity
continues
Environmental DNA protocol development guide for biomonitoring

20

Table 3

continued
Quality or stringency

Aspect ofproject

High (desirable)

Medium (minimum
requirements)Low(insufficient)

SampleUsing appropriate

Using appropriate

Equipment usedcollection

equipment andequipment andnot sufficientmethods to avoid

methods to avoid

to avoid

contamination,

contamination,

contamination,

conducted by staff

conducted by staff

staff with limitedwith relevant training

who have received

or no training

and expertisesufficient training

Quality

Positive and negative

Negative controls

assurance andcontrols during

during collection and controls

controlcollection, extraction

extraction

and processing

No or limited
throughout the
project

Analysis:

In-depth analysis

Reporting presence

ReportingqPCR

and validation ofor absence. Assay

presence orassays, reporting

may not be validated

absence only

analytical specificity,and may haveof detection. Samples

undergone minimum Assay testing
analytical sensitivity conditions areare analysed withand specificity

multiple technicaltesting. Samples are

and analyses

analysis includes

analysed using single are completed
in the absencetechnical replicates,

positive and negativeand each analysisof positive andsensitivity and limits

replicates, and each

not reported

controls to assess

includes positive and negative controls

method successnegative controls
to assess method
success

Analysis:

In-depth analysis,

Reporting presence

Reportingmetabarcoding

species richness

or absence andpresence or

estimates, occurrence

species richness

absence only

probability modelling,

estimates

error analysis
included
continues

Environmental DNA protocol development guide for biomonitoring

21

Table 3

continued
Quality or stringency

Aspect ofproject

Medium (minimum
requirements)Low(insufficient)

Some

No

communication with

communication with

communication

end users throughout

end users at initialwith end users

each phase of theand final phases

High (desirable)

Communication Clear and sustained

project
Optional extra

Parallel surveys with

Data available fromNo parallel

validation:

traditional methods

past parallel surveys

surveysparalleltargeting same taxa

traditional
surveys
Source: adapted from Jerde 2019.

Permissions and ethical considerations
Environmental DNA fieldwork might require research or collection permits,
depending on the place where samples are collected. Before the start of aneDNA project, project leaders should check with the relevant bodies to findout which permits are required. For example, some states require permits
to import samples from interstate, and research activities in national parks
require specific permits to be in place before the start of sample collecting.
Environmental DNA is a non-destructive sampling method (for largeorganisms), which negates the need for most of the human or animal ethic
approvals typical for other methods. At the time of writing, we are not awareof any institutions that require ethics approval for sampling eDNA fromwater, sediment, air or soil samples. We do, however, strongly recommend
consulting up-to-date regulations before commencing any new eDNA
research project.
Surveys will sometimes take place in culturally important locations or detect
culturally significant species. Co-design and co-implementation of projects
is important. First Nations peoples should be engaged during the project
design phase to ensure that methods consider cultural sensitivities andrelevant permissions are received (see Principle 6). Furthermore, there is achance that samples might inadvertently detect human DNA, which might beculturally sensitive for some Traditional Owner groups (Handsley-Davis et al.
2021). Honest and clear communication with Traditional Owners is essential
to avoid conflict and legal implications. In New Zealand, research practices
Environmental DNA protocol development guide for biomonitoring

22

should consider the Wai 262 principles (Waitangi Tribunal 2012). It is strongly
recommended that project outcomes are shared with Traditional Ownercommunities. The understanding of implications surrounding this issue isevolving and it is likely that new regulations will be developed in the future.
It is the responsibility of project leaders to stay informed and conform to
the most recent legislation on this matter. If potential concerns exist, it isadvisable to consult with experts at the start of a new eDNA project.

2

Collect samples

Environmental DNA can be collected from many environments andsubstrates, so sampling protocols can differ substantially between projects.
A few general principles should be followed when collecting eDNA samples.
The main considerations are collecting an adequate number of samples
for analysis, avoiding cross contamination, implementing appropriate
preservation methods, and ensuring an appropriate chain of custody.
Strict collection processes will preserve the integrity of your samples
and improve testing accuracy.
Logistics around sampling for eDNA can be complicated and should beconsidered early in the project. Topics include:• spatial and temporal variations in eDNA concentration in the environment(e.g. spawning events and migrations)• fieldwork conditions• sample collection method• available infrastructure at the sampling locations• training of collection teams.
Sampling should follow SOPs that are fit for purpose. Custom SOPs might
have to be designed to suit project needs or to ensure interoperability withother projects within the same organisation.

Collection process
The purpose of the research and experimental design will affect the collection
process. Different substrates, collection, and preservation methods requiredifferent field logistics and yield different results (Koziol et al. 2018). As such,they cannot be compared directly, which should be taken into consideration
for long-term or ongoing projects. Common substrates used to collect eDNA
include water, sediment, scat, and stomach content. However, eDNA can becollected from other substrates such as sweeping or vacuum samples, pollen

Environmental DNA protocol development guide for biomonitoring

23and grain. Preservation and collection methods will depend on available
resources and transport time to the laboratory.
In designing and describing the collection process, first establish the bestpractice approach to achieve the study purpose. Based on this best-practiceplan, describe the potential considerations and limitations specific to theproject. The sampling protocol should include a description of responsibility
for each step in the process, and a detailed description of preservation andstorage methods (see Preserve samples for processing).
Sample collection is often done by non-experts, including consultants
and community members who might be unfamiliar with the rigorous
contamination protocols required when collecting eDNA. In these cases,
the sampling plan should be designed for non-specialists, with full details
provided and written in non-technical language. Anticipate potential
problems in the sampling plan (e.g. a particular site is inaccessible, silty waterclogs filters) and provide backup plans to the sampling team to ensure that
useful data are still collected. Training videos or hands-on training may also
be necessary. If possible, test your sampling plan and training resources byrequesting non-technical staff to follow the instructions and identify which
points in the protocol cause confusion. Providing opportunities for feedback
from trainees and updating standard operating procedures accordingly canhelp to clarify the process.

Sample size
Deciding on a suitable sample size (number of samples and volume ofsamples collected) for a project will vary depending on the purpose of thestudy, the type of environment, the characteristics of the target species/
communities, and the known abundance of target species (Stauffer et al.
2021). The probability of detecting targeted DNA in any environment
depends on both the collection method and the concentration anddispersion of DNA at those sites (Furlan et al. 2019). However, several studies(e.g. Deiner et al. 2018; Li et al. 2018; Coutant et al. 2021) have shown thattargeted eDNA approaches can be quite robust to technical choices, withincommon ranges. The concentration of DNA in turn depends on the biology,
ecology and behaviour of the target species (e.g. shedding rates, mobility,
abundance) (Harrison et al. 2019).
Sampling frameworks can be based on available methods used to assess
eDNA assay sensitivity (Furlan et al. 2016) or published work on similar taxa
and environments. A pilot study may be needed to determine minimum
sample size for long-term monitoring projects, particularly in novel habitats
or with target taxa for which limited empirical eDNA data exist.

Environmental DNA protocol development guide for biomonitoring

24

Avoiding cross contamination
Reducing the risk of contamination should be a primary concern whencollecting samples, regardless of the type of sample being collected.
Common sources of contamination include environmental contaminationfrom other biological materials, poorly decontaminated sampling equipment,
and cross contamination between samples.
It is critical to establish clean and consistent field collection protocols that
reduce the probability of contamination. Decontamination of equipment
and other field gear prior to sampling events is essential for ensuring
sample independence (Goldberg et al. 2016). Consider single-use equipment
and containers for sampling, and clean and sterilise equipment between
sampling as well as before and after sampling. Decontaminating equipment
is best done using >3% sodium hypochlorite (bleach) to destroy any traces
of DNA/RNA. Field equipment supplies and field consumables must not be
stored in areas with high copy number DNA settings, such as PCR laboratoryor tissue handling areas.
Additional measures to reduce contamination risk should include diligent
use of gloves, presterilised equipment, avoiding potential sources ofcontamination (e.g. not interacting with other biological materials during
sampling), and including negative controls in the workflow.
Negative field controls (DNA-free samples collected using the same protocoland equipment, preserved and processed in the same way as field samples)
are critical for detecting contamination (Goldberg et al. 2016). Negative
controls should be collected before and after sample collection at each site
to identify potential sources of contamination.
Once collected, samples should be stored appropriately to avoid crosscontamination until they can be returned to the lab. Samples must not be
stored in areas with high copy number DNA settings, such as PCR laboratoryor tissue handling areas.
It should be noted that contamination can occur at any stage of the process.
Consistently implementing laboratory processes that reduce contamination
risks is therefore important. Such general processes can include• segregating laboratory activities based on DNA content and risk potential• introducing workflow automation where possible• choosing consumables that reduce contamination risk.

Environmental DNA protocol development guide for biomonitoring25

Chain of custody
The chain of custody links the samples collected in the environment to thefinal processing and analysis. A high-quality chain of custody is important
to ensure the final results of the study are valid and usable, particularly forlegal purposes.
A chain of custody record must be maintained. It must provide acomprehensive history of each sample. This includes how samples havebeen handled and transported (e.g. what temperatures samples were storedat) and who has been involved. Developing a reliable sample tracking system,
using barcodes or unique sample IDs, is key to doing this effectively.
During experimental design, define the chain of custody, including where it
starts, the data that should be recorded, and who is responsible. Throughout
the study, record all steps from sample collection through to processing andanalysis, including any variations away from the steps determined duringexperimental design.

Metadata and documentation
Collecting detailed metadata during sample collections will help to maintaina good chain of custody to support the analyses and interpretation of
results. This is especially relevant when different teams collect and analyse
data. The metadata scheme should be established before sample collection
begins and be provided to collection teams ahead of fieldwork (Table 4). If
possible, sampling kits can be prelabelled and templates provided for datacollection.
Standardised metadata following the FAIR principles (Findable, Accessible,
Interoperable, Reusable; Wilkinson et al. 2016) should be recorded acrossall projects. However, individual projects may have additional specific datathat must be collected. The use of standardised ontologies (e.g. Darwin Core)and metadata schemes and repositories developed for use with molecularsamples – such as GEOME – is highly recommended (Wieczorek et al. 2012,
Deck et al. 2017, Riginos et al. 2020).

Environmental DNA protocol development guide for biomonitoring

26

Table 4 Recommended metadata during sample collection
Metadata field

Notes

ExampleUnique sample

Clear code or designation of

eDNA-ProjCO6421, barcode

ID¹

sample unique to the sample

Name collector¹

Ideally including contact details Name Surname name.
surname@email.com+61412345678

Sample type¹

The type of sample that was

Water, scat, grain, bulk

collected
Date + time¹

Add estimated time of day if

07/04/2022, 15:00

exact time is not possible
Location¹

Site name and location

Battery Point, Hobart

GPS coordinates¹

Latitude and longitude−42.886201, 147.335827;

coordinates
Sample volume¹

Volume of water filtered,

42° 53’ 10’’ S, 147° 20’ 13’’ E
1 L, 10 gweight of sediment or bulk
sample collected
Collection

Method used to collect theFiltration method, sediment

method¹

samplecore, sweeping

Preservation

Method used to preserve

Freezing, drying, ethanol,

method¹

sampleLongmire’s solution

Project nameCan be a code or formal

eDNA standards project,designation

PR_eDNA_ST

Is sample a negative control or

Negative control

Control

not?
Habitat typeSpecific type of habitat

Coral reef, seamount, lake,
stream, forest, desert

Environmental

Environmental variables

Temperature (surface/at

conditions

relevant to the project aims

depth), weather conditions,or which might influence DNA

currents

concentrations
Other

Observations relevant to the

Fishing vessels nearby, coral

observations

project aims or which might

spawning, large storm priorinfluence DNA concentrations

to sampling
continues

Environmental DNA protocol development guide for biomonitoring

27

Table 4

continued

Metadata field

Notes

ExampleProblems

Issues that occurred during

Half of sample spilled, gloves

collecting that might

tore, filter clogged after 0.6 L

compromise sample integrity
Filter type¹ (ifType and pore size

used)

Polyethersulfone, cellulose
nitrate
0.2 μm, 1.2 μm

Collection depth

If sampling from water bodies

Surface, 10 m, 1200 m¹ indicates minimum required metadata

3

Preserve samples for processing

Environmental DNA and RNA begins decaying immediately after it isshed by an organism. DNA and RNA continue to degrade after they arecollected due to a range of factors, such as UV radiation, mechanical forces,
microbial activity, and spontaneous chemical reactions (Goldberg et al.
2016). Repeated freeze–thaw cycles of samples during or after preservation
can also cause significant DNA and RNA degradation. RNA is a less stablemolecule than DNA and usually degrades quicker than DNA. However,recent studies have demonstrated that decay-rate constants for eRNA vary,
but can be surprisingly similar to those of eDNA – environmental RNA canbe detected in water samples for 13–72 hours after production, and eRNA
concentrations can even be higher than corresponding eDNA (Yates et al.
2021).

Preservation of eDNA
To slow the degradation of samples, they should be preserved andprocessed using standardised protocols as part of the collection process(see Thomas et al. 2018), or as soon as possible after collection. Timeframes
for preservation and processing should be specified in the experimental
design. You may also need to consider whether long-term storage orarchiving of samples will be required. Different preservation methods
have different levels of handling involved, choosing a method that involves
minimal handling will reduce the risk of cross contamination.
For preservation, samples can be kept in cold conditions until processing
can occur (e.g. some samples can be kept in ice for up to 24 hours before
preserving without compromising detection; Pilliod et al. 2013), although
Environmental DNA protocol development guide for biomonitoring28

this depends on the conditions affecting samples at the time of collection.
In some cases, such as when collecting scat or soil samples, drying may be
a better preservation option, especially when maintaining a consistent cold
temperature is not possible. Drying can be achieved by using silica gel or
calcium chloride moisture-absorbing bags (Nagy 2010, Guerrieri et al. 2020).
Filtration methods can also be used, with the advantage that large volumesof water or air can be rapidly filtered and samples preserved onsite usingchemical preservation or dehydration (Thomas et al. 2019, Bruce et al. 2021).
However, this may be difficult to perform within a suitable timeframe if
appropriate sampling equipment is not available. If filtration is not practical,
precipitation (Ficetola et al. 2008) can be used to preserve small samples
by adding a salt (typically sodium acetate) and absolute ethanol in the field,
followed by storing the sample at −20 °C. However, precipitation is unwieldy
at large scale, and using ethanol and sodium acetate may enrich for large
DNA at the expense of small, degraded DNA.
Establishing backup samples by splitting a single sample into several canhelp to mitigate against contamination but may reduce the chance ofdetection if the target is at very low concentration. For DNA aliquoting, it isa good idea to freeze multiple aliquots to avoid multiple freeze–thaw cycles
during analysis.

Processing of eRNA
Extra care should be taken when targeting eRNA because RNA molecules
are even more vulnerable to degradation than DNA. RNA stability depends
strongly on the type of preservation in addition to sample collection,
harvesting, handling, transportation, and the extraction protocol used. The
gold standard for RNA preservation is immediate liquid nitrogen–based
cryopreservation (−80 °C) after flash freezing (Auer et al. 2014) or filtration;
however, this method may be problematic for many environmental sampling
applications that are without access to liquid nitrogen or suitable freezing
equipment. Alternatively, environmental samples can be submerged inaqueous, nontoxic stabilising solutions with a storage reagent that rapidly
permeates samples to stabilise and protect cellular RNA (e.g. RNALater,
Invitrogen). The samples must be cooled to −80 °C as early as possible andkept there until processing can occur.

Environmental DNA protocol development guide for biomonitoring

29

4

Extract and isolate DNA/RNA

Processes for extracting and isolating DNA/RNA should be based on
validated commercial extraction kits and methods that are widely available
and commonly used in eDNA-based research. Any novel extraction andisolation methods should be optimised and standardised for eDNA/RNA use.
Methods must be chosen according to the needs of the project and the typeof sample.

Extraction of eDNA
Commercial DNA extraction kits are available for different substrates,
including soils, biofilms, tissue and water. Soil DNA extraction kits, forexample, have been shown to be efficient at extracting DNA from manydifferent substrates (Hermans et al. 2018). eDNA recovery rates vary withpreservation and extraction methods (Hinlo et al. 2017). Furthermore,certain methods, such as precipitation methods, might not work well forsmall DNA fragments, so alternative methods should be considered when
expecting heavily degraded DNA.

Extraction of eRNA
Commercial RNA extraction kits, used in conjunction with DNase kits toensure no eDNA carryover, can be used to extract RNA from environmental
samples. If DNase is not completed following RNA extraction, then one such
step should be incorporated prior to reverse transcriptase of extracted
RNA. The field of eRNA testing is still young and extraction and isolation
methods are still being formally optimised and standardised for eRNA use.
Methods must be chosen according to the needs of the project and the typeof sample.

5 Ensure quality control and purificationof extracts
All eDNA/eRNA extracts that do not produce positive results must be
assessed for inhibitory substances (i.e. substances in a sample or extractthat have a negative effect on the PCR, reducing assay sensitivity andincreasing the risk of false negative results if they are not removed by thechosen extraction method). Processes should outline suitable methods toquantify total DNA/RNA yield and assess the presence of inhibiting factors.
Inhibition can be assessed during qPCR in 2 ways – using a dilution series

Environmental DNA protocol development guide for biomonitoring30

of the eDNA extract with at least 2 dilution points (neat and 1:10) or an
exogenous internal positive control.
In both cases, the presence of inhibitors is shown through evaluating
the cycle threshold (Ct) values. Specifically, diluted eDNA extracts free of
inhibition should behave quantitatively (i.e. 3.3 Ct shift between dilution
points), while inhibited samples will show delayed amplification (Bylemans
et al. 2017, Murray et al. 2015, White et al. 2020). In the same way, theexogenous internal positive control will also exhibit delayed amplificationin the presence of inhibitors (Conte et al. 2018, Furlan et al. 2015).
Some commercial DNA polymerase master mixes contain substances thatcan effectively counteract most qPCR inhibition from eDNA extracts (Janeet al. 2015), and commercial DNA extraction kits for complex substrates
are able to remove common inhibitors – these should be used byaccredited laboratories. When this is not possible, inhibitor removal steps
can also be included post-extraction to avoid false negative detections(Schrader et al. 2012).

Table 5 Types of controls that can be implemented during different stagesof environmental DNA workflows
When to use Control type

Purpose

MethodSampleNegative

DetectDuring fieldwork, collectcollection(= sampling blank)

contaminationa sample known to notduring sample

contain target DNA

collection(e.g. filter 1 L of milliQ
water)

Positive exogenous Detect whether

Add exogenous control

control

DNA collected in

to samples collected

the field amplifies

in the field. Single

well (e.g. presence

species (for qPCR),

of inhibitors)

or a real or synthetic
mock community formetabarcoding
continues

Environmental DNA protocol development guide for biomonitoring

31

Table 5continued

When to use Control type

Purpose

MethodDNA

Negative

DetectExtract a blank (no DNA)

extraction(= extraction blank) contamination

sampleduring extraction
Positive exogenous Detect issues incontrol

Add (non-target-

extraction process species) DNA to samples
collected in the field.
Single species (for qPCR),
or a real or synthetic
mock community formetabarcoding

PCR

Negative

Detect

Non-template technicalcontamination

replicates. Theseduring PCR set up

controls have all reaction
components, except
water replaces the target
template

Positive exogenous Detect inhibition

Add (non-target-species)

controlof extracts in PCR

DNA to eDNA extracts.

process

Single species (for qPCR),
or a real or synthetic
mock community formetabarcoding

Determine

Analyse environmental

method successextracts using a genericacross the wholeprimer assay designed toworkflow

amplify abundant, nontarget DNA that will besimultaneously sampled,
captured, extracted
and amplified (or only
amplified) with the target
species’ DNA

Source: adapted from Furlan & Gleeson 2016.

Environmental DNA protocol development guide for biomonitoring32The type of controls used depends on the project. Controls should be clearly
defined by the sampling process and other methods, such as molecularbiology and bioinformatics. Table 5 describes the range of possible controls.

6

Analyse extracts

There are several molecular methods being tested and explored for theirsuitability in testing environmental samples. These currently include theuse of digital PCR (Doi et al. 2015), loop-mediated isothermal amplification(LAMP; Williams et al. 2017), and high-throughput sequencing methods
coupled with metabarcoding techniques (Ruppert et al. 2019).
This section outlines the minimum requirements for authors, including:• description of the molecular method used to analyse environmental
samples• explanation of what is measured by the method (e.g. absorbance, droplet
ratio)• description of the appearance and criteria for:– positive results– negative results– indeterminate results– unreliable results.
As an example, we provide minimum requirements for quantitative PCR
assays used to test environmental samples.

Quantitative PCR analysis
For species-specific assays, it is recommended that eDNA extracts areanalysed and interpreted using probe-based quantitative PCR (qPCR) analysis
because it is more sensitive and specific than conventional PCR or SYBRbased qPCR. Probe-based qPCR assays use specific probes that are labelledwith a fluorescent dye (e.g. FAM, VIC, ABY or JUN) and a quencher (e.g. Minor
Groove Binding quenchers, or Black Hole Quenchers) in conjunction withforward and reverse primers. During qPCR, samples containing the target
eDNA bind the primer and probe and a fluorescent signal is measured in realtime during PCR amplification.

Environmental DNA protocol development guide for biomonitoring

33

A qPCR curve has 3 phases: the baseline, in which the reaction is only
beginning to occur and no fluorescence can yet be captured; the exponential
phase, in which the target DNA is ideally being doubled at every cycle; anda plateau phase, in which the reaction has exhausted amplification capacity
and capture of absorbance reaches a non-incremental phase (Figure 2).
Amplification of targeted sequences results in the measurement offluorescence that is directly proportional to the amount of doublestranded DNA present at each PCR cycle (Figure 2). Fluorescence in Applied
Biosystems real-time PCR software presents results as a semi-log graphusing the ΔRn, which is the magnitude of the signal generated by the given
set of PCR conditions (Figure 3).
Variance in amplification among technical replicates can occur due to low
template abundance in samples (Figure 4). Ideally, amplification of samples
should cross the common threshold with little variation (Figure 5). Evidenceof primer-dimer or early non-target amplification may be seen in the analysis(Figure 6).

Plateau

ar

0.75

Line

Relative ﬂuorescence

1.00

Ampliﬁcation threshold

0.50

0.25

0

Baseline
10

20

30

40

Cycle number
Source: modified from the SIGMA® Life Science qPCR technical Guide.

Figure 2 PCR amplification plots of relative fluorescence (standard X–Y plot)

Environmental DNA protocol development guide for biomonitoring

34

1E07

1E06

ΔRn

135,043.113596
1E05

1E04

1E03

5

10

15

20

25

30

35

40

45

50

55

Cycle
A

B

C

D

E

F

G

H

Amplification plots obtained using a 10-fold serial dilution of the same target (5 replicates of each level). Samples
with high concentrations of target template exhibit fluorescence in early cycles (e.g. red), while samples withlow concentrations of target template fluoresce in late cycles (e.g. blue). The horizontal red line is the common
fluorescence threshold of the analysis.

Figure 3 Probe-based quantitative PCR amplification plot

1E07

ΔRn

1E06

135,043.113596
1E05

1E04

1E03

5

10

15

20

25

30

35

40

45

50

55

Cycle
A

B

C

D

E

F

G

H

The horizontal red line is the common fluorescence threshold of the analysis.

Figure 4 Probe-based qPCR amplification plot of high (red) and low (purple)
concentrations of target DNA
Environmental DNA protocol development guide for biomonitoring

35

1E07

ΔRn

1E06

1E05

1E04

1E03

5

A

10

B

C

15

D

E

20

F

25

G

Cycle

30

35

40

45

50

55

H

Figure 5 Primer-dimer or non-target amplification in probe-based quantitative PCR
amplification plots
1E07

ΔRn

1E06

1E05

1E04

1E03

5

A

10

B

C

15

D

E

20

F

25

G

Cycle

30

35

40

45

50

55

H

Figure 6 Negative detection signal in probe-based quantitative PCR amplification plots

Environmental DNA protocol development guide for biomonitoring36

Positive detection
For positive results to be accepted as reliable, the following criteria arerecommended:• All positive controls must exhibit positive amplification curves that crossthe common fluorescence threshold at the expected cycle.
• All non-template controls, blank extraction and field controls should notshow amplification.
• Test sample PCR replicates exhibit positive amplification curves that cross
a common fluorescence threshold and are within Ct cut-off values that are
based on the limit of quantification and detection for each assay.
If these conditions are met, the results are assumed to be positive. To
confirm the positive result, amplicons must then be confirmed by Sanger
sequencing and show 99–100% pairwise similarity to accepted accessions in
online repositories.

Negative detection
For negative results to be accepted as reliable, the following criteria arerecommended:• All positive controls must exhibit positive amplification curves that crossthe common fluorescence threshold at the expected cycle.
• All non-template controls, blank extraction and field controls should notshow amplification.
• Test sample PCR replicates exhibit amplification curves that do not cross
a common fluorescence threshold or fall within Ct cut-off values that are
based on the limit of quantification and detection for each assay (Figure 6).

Indeterminate
PCR replicates are considered as indeterminate if:• all positive controls cross the common fluorescence threshold.
• all non-template controls and blank extraction and field controls do notshow amplification.
• test sample PCR replicates exhibit amplification curves that cross acommon fluorescence threshold outside of Ct cut-off values based on thelimit of quantification and detection for each assay.
If these conditions are met, the results are assumed to be indeterminate.
To confirm the indeterminate result, amplicons must then be confirmed
by Sanger sequencing and show 99–100% pairwise similarity to accepted
accessions in online repositories.
Environmental DNA protocol development guide for biomonitoring

37

Unreliable
Results are considered unreliable if they show any of the following
3 outcomes (Figure 7):• No positive controls show amplification.
• Non-template or blank controls show amplification.
• Field or extraction negative controls show amplification.
If this occurs, samples must be reanalysed with the following inclusions in
the plate:• Reaction replicates must be completed without template DNA or eDNA
to confirm reagent contamination. If any of these replicates crosses thecommon fluorescence threshold, then reagents used in the prior plate
are contaminated and should be disposed of immediately. Fresh stocks
of primer, probes, mastermix and water (and IPC, if used) should be usedfrom that point forward.
• Reaction replicates with extract from the extraction and field blanksamples. If any of these replicates crosses the common fluorescencethreshold, then cross contamination may have occurred during sample
collection (field controls) or during eDNA extraction processing (extraction
controls), and all analysed samples must be re-extracted. Samples shouldbe tested again.
If none of these replicates cross the common fluorescence threshold, thencross contamination may have occurred during the prior plate set up or
loading of positive controls and standard dilutions. Results from this plateshould be accepted instead of the previous results.

Environmental DNA protocol development guide for biomonitoring

38

qPCRresults(CTvalues)

Didfieldnegativecontrolsamplify?

Yes

Unreliableresultsduetofieldcrosscontamination

Yes

Unreliable
resultsdueto
extractioncross
contamination

Informprojectownerand
advisesuitableactionsNo
Didextraction
negativesamplify?

Isthereany
sample
left?

No

Yes

No
Didnon-template
controlsamplify?

Cleanareawith
bleachand
extractsamples
again.
RepeatqPCR
testing

Yes

Unreliableresults
duetoqPCRcross
contamination

Usefreshstocksand
consumables,cleanplateset
upareaandrepeatqPCRtest

No

Unreliableresults
duetoreaction
failureUsefreshpositivecontrol
stocksanddoublecheck
reactionmix.
RepeatqPCRtestNo
Didpositivecontrols
amplify?
Yes
Resultsarereliableandcanbeanalysed

Figure 7 Identifying unreliable results in qPCR analyses

Environmental DNA protocol development guide for biomonitoring

39

7Interpret and communicate results

Results of eDNA surveys can require a different approach to interpretationfrom traditional surveys. Target species detected in eDNA surveys might
not have been detected in previous or parallel surveys using traditional
methods. Such differences have raised concerns in the past (Sepulveda et al.
2020) and might need to be addressed. Typical differences between eDNA
metabarcoding methods and traditional methods investigating species
assemblages include detecting more cryptic and/or transient species. Thisis a reflection of normal traditional and eDNA method biases, rather thanerrors in the metabarcoding process.
When high levels of stringency are required, as can be the case in biosecurity
applications, addressing the level of certainty in positive or negative
detections of a target species might be crucial to inform management(Figure 8; see also Ensure good communication). Likewise, the potential for
false negative or positive results should be considered when interpreting
results. This approach ties in with explicitly considering the limitations
inherent to eDNA studies in general and those specific to the survey results.

ResultNodetections

Singlepositive
detection
Checkcontrolsandre-runanalysis

ActionNodetections

ResultPositive
detection(s)Collectmoresamplesatpositivesite(s)ActionNodetections

ResultActionMultiplepositive
detections

ContinueroutineDNA sampling

Singlepositive
detection

Multiplepositive
detections

IncreaseDNAsampling

Starttraditional
sampling

Bold arrow colours indicate risk of significant impacts; dark blue: low risk; green: medium risk; orange: high risk.

Figure 8 Example of a management decision tree for high-stringency, species-specificeDNA sampling results, such as for biosecurity projects

Environmental DNA protocol development guide for biomonitoring40

When communicating results, explain the meaning of results clearly andaccurately. Ensure any data visualisation does not distort meaning.
It is important to emphasise limitations and explain uncertainties(e.g. surrounding relative abundance data, taxonomic resolution, incomplete
reference libraries) to avoid end users misinterpreting data. This would likely
also include discussing the differences between false positives and negatives
and how they might relate to the purpose of the project. A clear way toreport negative results is describing them as ‘below detection level’ insteadof ‘zero’. Asking clients to voice their interpretation of results can help tocheck if they have correctly understood the meaning of important results.
The purpose of many eDNA applications is to inform management decisions.
While these decisions are unlikely to be taken by the team executing an
eDNA project, results of a study might require considering the options forfollow-up actions to provide clearer results, confirm positive and negative
detections, or address flaws in the study design.

Summary of key steps for environmental
DNA/RNA test protocols
Step 1 Experimental design:•

Define purpose•

Decide level of replication, spatial extent, assay•

Decide on appropriate analysis methods•

Consider fieldwork logistics•

Account for realistic budget

Step 2 Minimum protocols for collecting samples:•

Wear personal protective equipment (PPE), at minimum gloves
when collecting, take steps to avoid cross contamination•

Sterilise all equipment•

Include negative controls•

Record appropriate metadata

Environmental DNA protocol development guide for biomonitoring

41

Step 3 Minimum protocols for preserving samples forprocessing:•Specify the collection medium (e.g. filter mesh size, material, size)•

Specify the collection volume, container material•

Record the preservative details (e.g. concentration, hazardous
details)

Step 4 Minimum protocols to extract and isolate
DNA/RNA:•

Sterilise equipment and consumables (use single-use consumable
where possible)•

Use a validated extraction kit or method•

Perform an extraction of negative controls•

Include an endogenous control

Step 5 Minimum protocols to ensure quality control andpurification of extracts:•

Determine inhibition•

Include inhibitor removal steps and purification processes•

Determine final eDNA/eRNA extract yield and concentration

Step 6 Minimum protocols for analysis (this uses qPCR as
an example, but should be tailored to the selected
molecular technique):•Include positive and negative controls, including field andextraction negatives•

Record reaction concentrations and thermal profiles•

Include standard serial dilutions curve preparation and quality•

Record details on technical replicates and assay limit of detection•

Determine false positive amplification and cross contamination•

Determine non-target amplification•

Confirm positive detection

Step 7 Minimum protocols for interpretation andcommunication:•

Ensure interpretation takes the study purpose and stringency
needs into account•Communicate the results precisely and accurately, including
method limitations and levels of certainty

Environmental DNA protocol development guide for biomonitoring

42

Resources
Environmental eNA Test validation guidelines, Department of Agriculture,
Water and the Environment, Canberra.​
International best-practice guidelines:• European Union: Bruce K, Blackman R, Bourlat SJ, Hellström AM, Bakker
J, Bista I, Bohmann K, Bouchez A, Brys R, Clark K, Elbrecht V, Fazi S,
Fonseca V, Hänfling B, Leese F, Mächler E, Mahon AR, Meissner K, Panksep
K, Pawlowski J, Schmidt Yáñez P, Seymour M, Thalinger B, Valentini A,
Woodcock P, Traugott M, Vasselon V & Deiner K (2021) A practical guideto DNA-based methods for biodiversity assessment. Advanced Books,
doi:10.3897/ab.e68634.
• Japan: Minamoto T, Miya M, Sado T, Seino S, Doi H, Kondoh M, Nakamura
K, Takahara T, Yamamoto S, Yamanaka H, Araki H, Iwasaki W, Kasai A,
Masuda R & Uchii K (2021). An illustrated manual for environmental
DNA research: Water sampling guidelines and experimental protocols.
Environmental DNA 3(1):8–13, doi:10.1002/edn3.121.
• Switzerland: Pawlowski J, Apothéloz-Perret-Gentil L, Mächler E & Altermatt
F (2020). Environmental DNA applications in biomonitoring and bioassessmentof aquatic ecosystems: Guidelines. Federal Office for the Environment,
Bern, www.researchgate.net/publication/346511410_Environmental_
DNA_applications_for_biomonitoring_and_bioassessment_in_aquatic_
ecosystems.
Recent review papers:• Beng KC & Corlett RT (2020). Applications of environmental DNA(eDNA) in ecology and conservation: opportunities, challenges andprospects. Biodivers Conserv 29(7):2089–2121.
• Gaither MR, DiBattista JD, Leray M & von der Heyden S (2022).
Metabarcoding the marine environment: from single species tobiogeographic patterns. Environmental DNA 4(1):3–8.
• Pawlowski J, Bruce K, Panksep K, Aguirre FI, Amalfitano S, ApothlozPerret-Gentil L, Baussant T, Bouchez A, Carogati L, Cermakova K, CordierT, Corinaldesi C, Cost FO, Danovaro R, Dell’Anno A, Duarte S, Eisendle U,
Ferrari BJD, Frontalini F, Fruhe L, Haegerbaeumer A, Kisand V, Krolika A,
Lanzen A, Leese F, Lejzerowicz F, Lyautey E, Macek I, Sagova-Mareckova
M, Pearman JK, Pochon X, Stoeck T, Vivien R, Weigand A & Fazi S (2022).
Environmental DNA metabarcoding for benthic monitoring: A review
of sediment sampling and DNA extraction methods. Science of the Total
Environment 818:151783, doi:10.1016/j.scitotenv.2021.151783.

Environmental DNA protocol development guide for biomonitoring43• Rourke ML, Fowler AM, Hughes JM, Broadhurst MK, DiBattista JD, Fielder
S, Walburn JW & Furlan EM (2022). Environmental DNA (eDNA) as a tool forassessing fish biomass: A review of approaches and future considerations
for resource surveys. Environmental DNA 4(1):9–33.
• Yates MC, Derry AM & Cristescu ME (2021). Environmental RNA: arevolution in ecological resolution? Trends Ecol Evol 36(7):601–609,
doi:10.1016/j.tree.2021.03.001.
Relevant methods papers:• Calderón-Sanou I, Münkemüller T, Boyer F, Zinger L & Thuiller W (2020).
From environmental DNA sequences to ecological conclusions: How strongis the influence of methodological choices? J Biogeogr 47(1):93–206.
• Furlan EM, Gleeson D, Hardy CM & Duncan RP (2016). A framework for
estimating the sensitivity of eDNA surveys. Mol Ecol Resour 16(3):641–654,
doi:10.1111/1755-0998.12483.
• Kumar G, Eble JE & Gaither MR (2020). A practical guide to sample
preservation and pre-PCR processing of aquatic environmental
DNA. Mol Ecol Resour 20(1):29–39.
• Wood SA, Pochon X, Ming W, von Ammon U, Woods C, Carter M, Smith M,
Inglis G & Zaiko A (2019). Considerations for incorporating real-time PCR
assays into routine marine biosecurity surveillance programmes: a casestudy targeting the Mediterranean fanworm (Sabella spallanzanii) and clubtunicate (Styela clava). Genome, doi:10.1139/gen-2018-0021.
• Zaiko A, Greenfield P, Abbott C, von Ammon U, Bilewitch J, Bunce M,
Cristescu ME, Chariton A, Dowle E, Geller J, Ardura Gutierrez A, Hajibabaei
M, Haggard E, Inglis GJ, Lavery SD, Samuiloviene A, Simpson T, Stat M,
Stephenson S, Sutherland J, Thakur V, Westfall K, Wood SA, Wright M,
Zhang G & Pochon X (2021). Towards reproducible metabarcoding data:
Lessons from an international cross-laboratory experiment. Mol Ecol
Resour 22:519–538, doi:10.1111/1755-0998.13485• Zhang S, Zhao J & Yao M (2020). A comprehensive and comparativeevaluation of primers for metabarcoding eDNA from fish. Methods Ecology
Evol 11(12):1609–1625.
Other useful resources:• Atlas of Living Australia: Australia’s largest repository of biodiversity
occurrence data, including eDNA data
• Australian Microbiome Initiative: Characterising the diversity andecosystem services of microorganisms, includes lab and bioinformatic
workflows

Environmental DNA protocol development guide for biomonitoring

44

References
Auer H, Mobley J, Ayers L, Bowen J, Chuaqui R, Johnson LA, Livolsi VA, Lubensky IA,
McGarvey D, Monovich LC, Moskaluk CA, Rumpul CA, Sexton KC, Washington MK,
Wiles KR, Grizzle WE & Ramirez NC (2014). The effects of frozen tissue storage
conditions on the integrity of RNA and protein. Biotech Histochem 89, 518–528.
doi:10.3109/10520295.2014.904927.
Barnes MA, Chadderton WL, Jerde CL, Mahon AR, Turner CR & Lodge DM (2021).
Environmental conditions influence eDNA particle size distribution in aquatic
systems. Environmental DNA 3:643– 653, doi:10.1002/edn3.160.
Barnes MA, Turner CR, Jerde CL, Renshaw MA, Chadderton WL &Lodge DM (2014).
Environmental conditions influence eDNA persistence in aquatic systems.
Environ Sci Technol 48(3):1819–1827, doi:10.1021/es404734p.
Bruce K, Blackman R, Bourlat SJ, Hellström AM, Bakker J, Bista I, Bohmann K,
Bouchez A, Brys R, Clark K, Elbrecht V, Fazi S, Fonseca V, Hänfling B, Leese F,
Mächler E, Mahon AR, Meissner K, Panksep K, Pawlowski J, Schmidt Yáñez P,
Seymour M, Thalinger B, Valentini A, Woodcock P, Traugott M, Vasselon V &Deiner K (2021). A practical guide to DNA-based methods for biodiversity assessment.
Advanced Books, doi:10.3897/ab.e68634.
Coutant O, Cantera I, Cilleros K, Dejean T, Valentini A, Murienne J & Brosse S (2021).
Detecting fish assemblages with environmental DNA: does protocol matter?
Testing eDNA metabarcoding method robustness. Environmental DNA 3 (3):619–30.
DAWE (Australian Government Department of Agriculture, Water and theEnvironment) (2021). Guide for authors writing an ANZSDP, www.agriculture.gov.
au/animal/health/laboratories/procedures/anzsdp-reviews/author-guidelines.
Deck J, Gaither MR, Ewing R, Bird CE, Davies N, Meyer C, Riginos C, Toonen RJ &Crandall ED (2017). The Genomic Observatories Metadatabase (GeOMe): A new
repository for field and sampling event metadata associated with genetic
samples. PLoS Biology 15(8):e2002925, doi:10.1371/journal.pbio.2002925.
Deiner K, Bik HM, Mächler E, Seymor M, Lacoursière-Roussel A, Altermatt F,
Creer S, Bista I, Lodge DM, de Vere N, Pfrender ME & Bernatchez L (2015).
Environmental DNA metabarcoding: Transforming how we survey animal andplant communities. Mol Ecol 26(21):5872–5895, doi:10.1111/mec.14350.

Environmental DNA protocol development guide for biomonitoring

45

Deiner K, Lopez J, Bourne S, Holman L, Seymour M, Grey EK, Lacoursière A, Li Y,
Renshaw MA, Pfrender ME, Rius M, Bernatchez L & Lodge DM (2018). Optimising
the detection of marine taxonomic richness using environmental DNA
metabarcoding: the effects of filter material, pore size and extraction method.
Metabarcoding and Metagenomics 2 (February):e28963.
Doi H, Uchii K, Takahara T, Matsuhashi S, Yamanaka H & Minomoto T(2015), Use of droplet digital PCR for Estimation of fish abundanceand biomass in environmental DNA survey. PloS One 10(3): e0122763,
doi:10.1371/journal.pone.0122763.
Furlan EM & Gleeson D (2016) Improving reliability in environmental DNA detection
surveys through enhanced quality control. Marine and Freshwater Research
68:388-395, doi:10.1071/MF15349.
Furlan EM, Gleeson D, Hardy CM & Duncan RP (2016). A framework for
estimating the sensitivity of eDNA surveys. Mol Ecol Resour 16(3):641–654,
doi:10.1111/1755-0998.12483.
Furlan EM, Gleeson D, Wisniewski C, Yick J, Duncan RP & Strecker A (2019). eDNA
surveys to detect species at very low densities: A case study of European
carp eradication in Tasmania, Australia. The J Appl Ecol 56(11):2505–2517,
doi:10.1111/1365-2664.13485.
Guerrieri A, Bonin A, Münkemüller T, Gielly L, Thuiller W & Francesco Ficetola G(2021). Effects of soil preservation for biodiversity monitoring using
environmental DNA. Mol Ecol 30: 3313–3325, doi:10.1111/mec.15674.
Goldberg CS, Turner CR, Deiner K, Klymus KE, Thomsen PF, Murphy MA, Spear SF,
McKee A, Oyler-McCance SJ, Cornman RS, Laramie MB, Mahon AR, Lance RF,
Pilliod DS, Strickler KM, Waits LP, Fremier AK, Takahara T, Herder JE & Taberlet
P (2016). Critical considerations for the application of environmental DNA
methods to detect aquatic species. Methods Ecol Evol 7(11):1299–1307,
doi:10.1111/2041-210X.12595.
Handsley-Davis M, Kowal E, Russell L & Weyrish LS (2021). Researchers using
environmental DNA must engage ethically with Indigenous communities. Nature
Ecology and Evolution 5(2):146–148.
Harrison JB, Sunday JM & Rogers SM (2019). Predicting the fate of eDNA in theenvironment and implications for studying biodiversity. Proceedings of the Royal
Society B 286, 191519:20191409.
Hermans SM, Buckley HL & Lear G (2018). Optimal extraction methods for thesimultaneous analysis of DNA from diverse organisms and sample type. Mol EcolRes 18(3):557–569, doi:10.1111/1755-0998.12762.
Environmental DNA protocol development guide for biomonitoring46

Hinlo GD, Lintermans M & Furlan E (2017). Methods to maximise recovery ofenvironmental DNA from water samples. PloS One 12(6), e0179251–e0179251,
doi:10.1371/journal.pone.0179251.
IPPC (International Plant Protection Convention) (2016). International Standard for
Phytosanitary measures 27: Diagnostic protocols for regulated pests. In: Food andAgriculture Organization of the United Nations: International Plant Protection
Convention, accessed 18 June 2021, ippc.int/static/media/files/publication/
en/2016/01/ISPM_27_2006_WithoutApp2_En_2015-12-22_PostCPM10_
InkAmReformatted.pdf.
Jerde CL (2021). Can we manage fisheries with the inherent uncertainty from eDNA?
J Fish Biol 98(2):341–353.
Jo T, Murakami H, Masuda R, Sakata MK, Yamamoto S & Minamoto T (2017).
Rapid degradation of longer DNA fragments enables the improved
estimation of distribution and biomass using environmental DNA. Mol Ecol
Resour 17(6):e25–e33, doi:10.1111/1755-0998.12685.
Klymus KE, Merkes CM, Allison MJ, Goldberg CS, Helbing CC, Hunter ME, Jackson CA,
Lance RF, Mangan AM, Monroe EM, Piaggio AJ, Stokdyk JP, Wilson CC & Richter
CA (2020). Reporting the limits of detection (LOD) and quantification (LOQ) for
environmental DNA assays. Environmental DNA 2:271–282, doi:10.1002/edn3.29.
Langlois VS, Allison MJ, Bergman LC, To TA & Helbing CC (2021). The need for robustqPCR-based eDNA detection assays in environmental monitoring and species
inventories. Environmental DNA 3(3):519–527, doi:10.1002/edn3.164.
Li J, Lawson Handley LJ, Read DS & Hänfling B (2018). the effect of filtration
method on the efficiency of environmental DNA capture and quantification via
metabarcoding. Mol Ecol Resour 18:1102–1114, doi:10.1111/1755-0998.12899.
Minamoto T, Miya M, Sado T, Seino S, Doi H, Kondoh M, Nakamura K, Takahara T,
Yamamoto S, Yamanaka H, Araki H, Iwasaki W, Kasai A, Masuda R & Uchii
K (2021). An illustrated manual for environmental DNA research: Water
sampling guidelines and experimental protocols. Environmental DNA 3(1):8–13,
doi:10.1002/edn3.121.
Nagy ZT (2010). A hands-on overview of tissue preservation methods for molecular
genetic analyses. Org Divers Evol 10:91–105, doi:10.1007/s13127-010-0012-4.
OIE (World Organisation for Animal Health) (2021). Manual of diagnostic tests
and vaccines for terrestrial animals 2021, OIE, Paris, accessed 15 June 2021,
oie.int/en/what-we-do/standards/codes-and-manuals/terrestrial-manual-online-access.

Environmental DNA protocol development guide for biomonitoring

47

Pawlowski J, Apothéloz-Perret-Gentil L, Mächler E & Altermatt F (2020). Environmental
DNA applications in biomonitoring and bioassessment of aquatic ecosystems:
Guidelines. Federal Office for the Environment, Bern, researchgate.net/
publication/346511410_Environmental_DNA_applications_for_biomonitoring_
and_bioassessment_in_aquatic_ecosystems.
Pokon X, Ziako A, Fletcher Lm, Laroche O & Wood SA (2017). Wanted dead or
alive? Using metabaroding of environmental DNA and RNA to distinguishliving assemblages for biosecurity applications. PloS One 12(11):e0187636,
doi:10.1371/journal.pone.0187636.
Riginos C, Crandall ED, Liggins L, Gaither MR, Ewing RB, Meyer C, Andrews KR,
Euclide PT, Titus BM, Overgaard Therkildsen N, Salces-Castellano A, Stewart LC,
Toonen RJ & Deck J (2020). Building a global genomics observatory: Using GEOME(the Genomic Observatories Metadatabase) to expedite and improve deposition
and retrieval of genetic data and metadata for biodiversity research Mol Ecol
Resour 20(6):1458–1469, doi:10.1111/1755-0998.13269.
Ruppert KM, Kline RJ & Rahman MS (2019). Past, present, and future perspectivesof environmental DNA (eDNA) metabarcoding: A systematic review in methods,
monitoring, and applications of global eDNA. Glob Ecol Conserv 17:e00547,
doi:10.1016/j.gecco.2019.e00547.
Salomé Stauffer S, Jucker M, Keggin T, Marques V, Andrello M, Bessudo S,
Cheutin M-C, Borrero-Pérez GH, Richards E, Dejean T, Hocdé R, Juhel J-B, Ladino F,
Letessier TB, Loiseau N, Maire E, Mouillot D, Martinezguerra MM, Manel S,
Fernández AP, Valentini A, Velez L, Albouy C, Pellissier L & Waldock C (2021). How
many replicates to accurately estimate fish biodiversity using environmental DNA
on coral reefs? Ecology and Evolution 11(21):14630–14643, doi:10.1002/ece3.8150.
SPHD (Subcommittee on Plant Health Diagnostics) (2019). Development of National
Diagnostic Protocols – Procedures for Authors Reference Standard (SPHD RS
No.2), National Biosecurity Diagnostic Network, Plant Health Australia, accessed
31 August 2021, plantbiosecuritydiagnostics.net.au/initiatives/nationaldiagnostic-protocols.
Stoeckle BC, Beggel, S, Cerwenka AF, Motivans E, Kuehn R & Geist J (2017).
A systematic approach to evaluate the influence of environmental
conditions on eDNA detection success in aquatic ecosystems. PloS
One 12(12):e0189119–e0189119, doi:10.1371/journal.pone.0189119.
Thalinger B, Deiner K, Harper LR, Rees HC, Blackman RC, Sint D, Traugott M,
Goldberg CS & Bruce K (2021). A validation scale to determine the readinessof environmental DNA assays for routine species monitoring. Environmental
DNA 3(4):823–836, doi:10.1002/edn3.189.

Environmental DNA protocol development guide for biomonitoring

48

Thomas AC, Howard J, Nguyen PL, Seimon TA & Goldberg CS (2018). eDNA
Sampler: A fully integrated environmental DNA sampling system. Methods Ecol
Evol 9(6):1379–1385, doi:10.1111/2041-210X.12994.
Thomas AC, Nguyen PL, Howard J & Goldberg CS (2019) A self-preserving, partiallybiodegradable eDNA Filter. Methods Ecol Evol 10(8):1136–1141.
Waitangi Tribunal Report 2012. The stage 1 report on the national freshwater andgeothermal resources claim. Lower Hutt, Legislation Direct. p258, https://forms.
justice.govt.nz/search/Documents/WT/wt_DOC_59941926/Wai2358W.pdf.
Wieczorek J, Bloom D, Guralnick R, Blum S, Doring M, Giovanni R, Robertson T &Vieglais D (2012). Darwin Core: an evolving community-developed biodiversity
data standard. PloS One 7(1):e29715, doi:10.1371/journal.pone.0029715.
Wilkinson MD, Dumontier M, Aalbersberg IJ, Appleton G, Axton M, Baak A,
Blomberg N, Boiten JW, da Silva Santos LB, Bourne PE, Bouwman J,
Brookes AJ, Clark T, Crosas M, Dillo I, Dumon O, Edmunds S, Evelo CT,
Finkers R, Gonzalez-Beltran A, Gray AJG, Groth P, Goble C, Grethe JS,
Heringa J, ’t Hoen PAC, Hooft R, Kuhn T, Kok R, Kok J, Lusher SJ, Martone ME,
Mons A, Packer AL, Persson B, Rocca-Serra P, Roos M, van Schaik R,
Sansone S-A, Schultes E, Sengstag T, Slater T, Strawn G, Swertz MA,
Thompson M, van der Lei J, van Mulligen E, Velterop J, Waagmeester A,
Wittenburg P, Wolstencroft K, Zhao J & Mons B (2016). The FAIR Guiding
Principles for scientific data management and stewardship. ScientificData 3:160018, doi:10.1038/sdata.2016.18.
Williams MR, Stedtfeld RD, Engle C, Salach P, Fakher U, Stedtfeld T, Dreelin E,
Stevenson RJ, Latimore J & Hashsham SA et al. (2017). Isothermal
amplification of environmental DNA (eDNA) for direct field-based monitoring
and laboratory confirmation of Dreissena sp. PloS One 12(10):e0186462,
doi:10.1371/journal.pone.0186462.
Zaiko A, Greenfield P, Abbott C, von Ammon U, Bilewitch J, Bunce M, Cristescu ME,
Chariton A, Dowle E, Geller J, Ardura Gutierrez A, Hajibabaei M, Haggard E,
Inglis GJ, Lavery SD, Samuiloviene A, Simpson T, Stat M, Stephenson S,
Sutherland J, Thakur V, Westfall K, Wood SA, Wright M, Zhang G &Pochon X (2021). Towards reproducible metabarcoding data: Lessons froman international cross-laboratory experiment. Mol Ecol Resour 22:519–538,
doi:10.1111/1755-0998.13485.

Environmental DNA protocol development guide for biomonitoring49