Environmental DNA test validation guidelines

Abstract

The use of molecular assays to assess the presence of species using environmental DNA (eDNA) and RNA (eRNA) as analytes has diversified as the field of environmental surveillance advances. These methods can be used, for example, to screen for pest species as part of biosecurity measures and risk management, to screen for threatened species as part of development requirements, or for biodiversity monitoring purposes. To ensure that testing results are reliable, it is imperative to develop and validate molecular assays using
stringent quality standards that can minimise the potential for false negative and false positive results.

Full text

Environmental
DNA test
validation
guidelines

© University of Canberra 2022
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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 test
validation guidelines.National eDNA Reference Centre, Canberra.
ISBN 978-1-74088-533-1
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 this
publication 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 from
across 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 as
knowledge broker.
Editing and design: Biotext Pty Ltd

Contents
Glossary  5
Introduction  7
Assaypurposeandselection  10
Species-specicassaydevelopmentandvalidation  12
1 Dene the intended purpose of the assay 13
2 Design and test the assay 13
3 Validate and optimise the assay using reference samples 15
4 Check analytical specicity 16
5 Check analytical sensitivity 17
6 Check repeatability 17
7 Check reproducibility 18
8 Determine thresholds (cut-os) 19
Summary of key steps in species-specic qPCR assay development
and validation 19
Resources 21

Metabarcodingassaydevelopmentandvalidation  22
1 Dene the intended purpose of the assay 23
2 Design and test the assay 23
3 Validate and optimise the assay using reference samples 27
4 Check reproducibility 28
Summary of key steps in metabarcoding assay development and
validation 28
Resources 29
References  30

Environmental DNA test validation guidelines 5
Glossary
Assay: The laboratory workow from DNA extraction to sequence outputs.
Often refers to the target gene and taxonomic group (e.g.16S_Fish,
18Suniversal, COI).
Endogenous control: 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 a positive control to monitor method success.
Exogenous DNA templates can be generated from custom-synthesised DNA
fragments, DNA extract, plasmids, competent cells or viral particulates, and
be added to samples during any stage of the eDNA workow after sample
collection. Endogenous controls use the fact that DNA is ubiquitous in the
environment, such that every environmental sample will contain DNA from
multiple sources. In this context, a generic primer assay can be designed
to amplify abundant, non-target DNA that will be simultaneously sampled,
captured, extracted and amplied with the target species DNA (Furlan &
Gleeson 2016).
Environmental DNA/RNA (eDNA/eRNA): DNA or RNA directly extracted
from environmental samples (e.g.soil, sediment, water) without any
knowledge of the original organism. DNA carries genetic information,
whereas RNA transfers information to produce specic proteins and is only
shed by physiologically active (living) organisms.
Haplotype: A group of alleles that are inherited together from a single
parent – for example, mitochondrial haplotypes. A haplogroup consists
of haplotypes that shared a common ancestor with a single nucleotide
polymorphism mutation.
High-throughput sequencing (HTS): A technique able to determine the
nucleotide composition of millions of nucleic acid sequences. Dierent
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).
HTS library: Amplicons amplied for the purpose of HTS, cleaned and
pooled following indexing.
Inhibitory substances: Substances in a sample or extract that have a
negative eect on PCR, reducing assay sensitivity and increasing the risk of
false negative results.

Environmental DNA test validation guidelines 6
Limit of detection (LOD): The lowest concentration of target DNA that can
be detected with a dened level of condence (usually 95% detection rate).
Limit of quantication (LOQ): The lowest amount of DNA in a sample
that can be quantitatively determined with a stated precision, under stated
experimental conditions.
Metabarcoding: Simultaneous taxonomic identication of Operational
Taxonomic Units (OTUs) or Amplicon Specic Variances (ASVs) in eDNA
samples with millions of sequences, generated by PCR amplication using
one of the HTS techniques.
Monitoring: Systematic collection of data over time to detect changes in
a system (Gerber etal. 2005). Data can include information on a range of
factors, such as environmental, ecological, biological and social.
Polymerase chain reaction (PCR): A molecular technique that allows
exponential amplication of a target fragment/region of DNA from a mixture
of DNA fragments. The desired fragment to amplify is recognised from the
other fragments in the mixture by specic primers (small single-stranded
oligonucleotides) complementary to the desired sequence.
Primer: Short DNA fragments used in PCR amplication that bind adjacent
to the target region/gene.
Quantitative PCR (qPCR): A variant of PCR. The main dierence between
the two is that qPCR can quantify how many fragments of DNA are amplied
during each step in the reaction, leading to quantitative data.
Reference library: Database with DNA barcodes of specic species.
Sequencing: Determining the order of nucleotides in DNA or RNA; this can
be done using various methods.

Environmental DNA test validation guidelines 7
Introduction
The use of molecular assays to assess the presence of species
using environmental DNA (eDNA) and RNA (eRNA) as analytes
has diversied as the eld of environmental surveillance
advances. These methods can be used, for example, to
screen for pest species as part of biosecurity measures and
risk management, to screen for threatened species as part
of development requirements, or for biodiversity monitoring
purposes. To ensure that testing results are reliable, it is
imperative to develop and validate molecular assays using
stringent quality standards that can minimise the potential for
false negative and false positive results.
What is the aim of these guidelines?
These Environmental DNA test validation guidelines provide harmonised quality
control and minimum standard considerations for developing or validating
eDNA/eRNA assays for the purpose of single-species or multi-species
detection.
This document is a comprehensive guide for the development and use
of eDNA/eRNA tests, recommended and curated by eDNA experts,
stakeholders and end users in Australia and New Zealand. The guidelines
are designed to support a consistent and best-practice approach to eDNA/
eRNA testing to help detect species of interest. This approach ensures that
surveillance and resource managers are provided with robust scientic
evidence to support decision making.

Environmental DNA test validation guidelines 8
Environmental DNA–based methods exist for a broad variety of organisms;
however, assay performance has only been supercially tested for most
available assays, and few assays are validated to ensure reproducibility
(Thalinger etal. 2021). This document outlines minimal quality requirements
to develop and validate eDNA/eRNA molecular assays and standard
protocols for operational use in Australia and New Zealand.
Who are they for?
The Environmental DNA test validation guidelines provide minimum quality
requirements and assurances to design and validate eDNA assays for
biosecurity and surveillance applications. Although these guidelines will help
improve the accuracy and reliability of eDNA assays, they are not explicitly
designed to provide results for use in compliance and legal situations.
For researchers
The Environmental DNA test validation guidelines detail key steps to be
used in assay development and validation for species-specic testing
andmetabarcoding.
For clients
The Environmental DNA test validation guidelines provide quality assurance for
any contracted eDNA work. They tell end users what services and standards
can be expected, and may also be used to inform sta collecting samples or
involved in other areas of the project.
How have they been developed?
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 Standards and Best Practices Committee of
the Southern eDNA Society, 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 ocials and end users were held in 2021–22 to
ensure that the guidelines were t for purpose and met the highest quality
standards in the eld.

Environmental DNA test validation guidelines 9
Guidelines for single-species detection assays use a framework based
on the current principles and methods of validation for diagnostic
assays as approved by the World Organisation for Animal Health and the
International Standard for Phytosanitary Measures from the International
Plant Protection Convention. These principles and methods dene the
minimum requirements needed to determine the tness of an assay,
including analytical sensitivity, assay specicity, limit of detection, limit of
quantication, and eDNA-based ex situ testing.
Guidelines for multi-species detection assays use a framework based on
current recommendations from existing eDNA metabarcoding guidelines,
including the Environmental DNA sampling and experiment manual from the
Japanese eDNA Society (Minamoto etal. 2021), A practical guide to DNA-based
methods for biodiversity assessment (Bruce etal. 2021, Zaiko etal. 2021), and
existing protocols from experts in Australia and New Zealand.
Updates
It is anticipated that the Environmental DNA test validation guidelines will be
updated and expanded over time, with 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 eld of
environmental DNA.

Environmental DNA test validation guidelines 10
Assay purpose and selection
It is important that the intended purpose of an assay is dened
at the outset. This will help to determine whether a species-
specic, metabarcoding or combined approach is appropriate.
The characteristics of the assay can also be checked against its
purpose throughout the development and validation process,
to ensure that the assay is t for purpose. This ensures that
eDNA and eRNA assays and related procedures are appropriate
and results are relevant to management.
Environmental DNA assay performance is aected by
environmental factors that must be considered for assay
optimisation. After initial optimisation for an intended purpose,
characteristics of the performance of the assay must be tested
and checked against the intended purpose and overall project
principles.
The most common purposes of species-specic eDNA/eRNA assays (see
Species-specic assay development and validation) are to:
• contribute to the detection of a targeted species in
–an environment
–waste products of a host organism
–a compartment or storage area
• assess the presence of metabolically active species in
–an environment
–waste products of a host organism
–a compartment or storage area
• contribute to the detection and eradication of invasive species and pests
in dened areas.

Environmental DNA test validation guidelines 11
The most common purposes of metabarcoding eDNA/eRNA assays (see
Metabarcoding assay development and validation) are to:
• characterise and survey biodiversity in
–an environment
–waste products of a host organism
–stomach contents of an organism
• contribute to the monitoring of species assemblages
• contribute to the detection and eradication of multiple invasive species
and pests in dened areas.
For both species-specic assays and metabarcoding, there may be many
more specic purposes for which assays can be developed. Such specic
applications and their unique purposes need to be clearly dened within the
context of a fully validated assay.
In addition, consider whether a combined approach may be useful. For
example, you may decide to include metabarcoding as your rst step in a
screening tool to determine the range of species at the test site, to then
guide the use of more targeted, species-specic assays.

Environmental DNA test validation guidelines 12
Species-specic assay
development and validation
Species-specic eDNA assays are currently PCR-based assays
that involve amplication of a target DNA sequence present in
environmental matrices, and can be validated and designed
within a diagnostic framework. However, the availability of
suitable eDNA samples and experimental set-ups to formally
assess diagnostic sensitivity and specicity is rare. Validating
eDNA/eRNA assays for the purpose of diagnostic assessments
requires a quantitative analysis of amplication data with
minimal variation to conrm diagnostic capability. Therefore,
the robustness of eDNA/eRNA assays must be considered and
explored extensively todetermine how inhibiting factors and
environmental factors aect the amplication of known eDNA/
eRNA tracesincontrolledmatrices.
Steps for species-specic assay development and validation are:
1 Dene the intended purpose of the assay
2 Design and test the assay
3 Validate and optimise the assay using reference samples
4 Check analytical specicity
5 Check analytical sensitivity
6 Check repeatability
7 Check reproducibility
8 Determine thresholds (cut-os)

Environmental DNA test validation guidelines 13
1 Dene the intended purpose of the assay
The purpose of the assay must be dened at the outset, to act as a
benchmark for testing and validation (see Assay purpose and selection).
The purpose should be dened in terms of:
• the high-level purpose (e.g.to detect the target species in a certain area,
to contribute to managing invasive species in an area)
• the target organism
• what aspect of the target organism will be measured (e.g.presence of live
organisms, presence of excreta, presence of cells)
• the sampling matrix.
2 Design and test the assay
The robustness of eDNA detection relies heavily on the design quality
of primers and probes (Langlois etal. 2021). Species-specic assays are
recommended to be designed using probe-based chemistries for species-
specic detection.
Assays must be designed and tested in accordance with the assay’s intended
purpose.
Assay choice or design
The rst step is to ascertain whether tests have already been developed for
the intended purpose or are available for testing. Suitable molecular assays
may have been designed and validated for purposes outside the scope
of eDNA testing. In the specic case of biosecurity applications, national
diagnostic protocols may be available for targeted species with approved
molecular assays that could be suitable for eDNA-based testing.
When designing or testing an eDNA/eRNA assay, DNA degradation and
inhibition must be considered as important factors that aect assay
performance. Environmental DNA/RNA assays are designed considering
environmental factors that aect DNA integrity and the success of detection.
Such factors include temperature, moisture, bacterial activity and UV
radiation, which aect DNA degradation rates. Natural inhibitors such as
algae, humic substances and suspended sediment particles can also aect
detection success (Stoeckle etal. 2017).

Environmental DNA test validation guidelines 14
Therefore, primers and probes are commonly designed to target short
(80–280base pairs) rather than large (>300base pairs) fragments, because
short fragments remain available for detection for longer (Jo etal. 2017).
Targeted regions should be selected following currently accepted barcode
regions, and considering the availability of reference DNA data for the
species.
In silico testing
If relevant sequence data for the target species (and related non-target
species) exist in online repositories, selected assays should undergo in
silicotesting for specicity using sequence alignment tools (e.g.BLAST).
Common repositories include the National Center for Biotechnology
Information, the Barcode of Life Data System, the European Molecular
Biology Laboratory online repositories, and the CSIRO National Biodiversity
DNA Library.
If such data do not exist, reference material will need to be obtained and
sequenced to facilitate assay design. The level of specicity depends on
the purpose of the assay; however, assays should assess the specicity of
the target species, considering potential intraspecic variation in the study
area (i.e.inclusivity), as well as non-amplication of co-occurring and closely
related species (i.e.exclusivity). The biology and ecology of the target species
should be considered – genetically diverse species will require greater
reference resources to achieve a similar level of assay robustness to that of
less diverse species.
Species-specic primers should be designed to display 0 base pair
mismatches with the target species. Probes are recommended to be
designed to display >2mismatches with closely related species (Klymus
etal. 2020a). If target species are part of a species complex or if the target
has minimal genetic dierences from closely related species, the use of
amplication-refractory mutation system primers and locked nucleic acid
probes is recommended to increase assay sensitivity and specicity of
annealing temperatures (Stewart etal. 2016). Testing for specicity should
comprise closely related non-target species, as well as commonly co-habiting
species that users could encounter in the environment. Suitable species and
samples for assay evaluation should be selected considering:
• taxonomy – include closest relatives (e.g.all members of the same genus
or family, and members of all suspected subspecies/genetically distinct
populations of the target species)

Environmental DNA test validation guidelines 15
• geographic scope of intended use – include representatives of all taxa
within the broader group (e.g.mammals) that might be encountered in the
study area.
Inhibition testing
The robustness of eDNA assays must also be tested against inhibitory
factors that interfere with assay performance. Assessment of robustness
against inhibition should begin during the assay development and
optimisation stages. The factors most likely to aect assay robustness
include pH, temperature and organic matrix factors (Goldberg etal. 2016).
If you have access to suitable environmental reference samples (seeStep3),
testing for inhibitory factors should be performed to conrm which factors
are present and in what concentrations following extraction, and whether
selected extraction methods are sucient to yield high-quality DNA/
RNA. Inhibition tests can then be undertaken using serial dilutions of
conrmed inhibitory matrices spiked with known concentrations of synthetic
oligonucleotides that reproduce the sequence of the qPCR assay target
sequence. Synthetic oligonucleotides can include nucleotide inversions
to allow for additional plate preparation controls to determine potential
cross-contamination of synthetic oligonucleotides (Trujillo-González etal.
2021). Assays would be considered robust if 10fold serial dilutions of the
synthetic oligonucleotides amplify within 3.3cycles ± standard deviation
(95% condence intervals) of each other within serial dilutions of inhibitory
matrices. Instances where amplication falls outside this acceptable
range would indicate lack of robustness in the assessed inhibitory matrix
concentration.
3 Validate and optimise the assay using
reference samples
Optimisation aims to evaluate and adjust the most important physical,
chemical and biological parameters of an assay to ensure that the
performance characteristics of the assay are best suited to the intended
application (OIE 2021). For analysing environmental samples, it is important
to select reference samples that are representative of the target species
and analyte. These reference samples may be uids, tissues, excreta and
environmental samples that contain the analyte of interest and are usually
harvested from the target species as well as their environments. Assays
should be optimised using both sets of reference samples (i.e.samples
derived from the target species and its environment).

Environmental DNA test validation guidelines 16
Environmental reference samples are considered to be samples known to
contain the analyte of interest in varying concentrations (modied from OIE
2021, Chapter 2.2.6: Selection and use of reference samples and panels).
They can ideally be collected from ex situ settings used to maintain the
target species; however, samples collected from natural environments
or urban locations with conrmed records of the target species may also
serve as suitable conrmed environmental samples. Sequences could be
obtained from the study area to account for potential genetic variation;
however, this may only be possible in locations where the target species is
already present in the environment. It is not a viable option for biosecurity
applications where target species can be considered exotic or their presence
is unknown. The suitability of environmental samples for assay optimisation
must be assessed based on the size of the habitat, the abundance of the
target species (Goldberg etal. 2016) and the availability of samples to be
appropriately standardised.
The availability of conrmed environmental samples may be limited,
depending on the target species. Reference environmental samples spiked
using extracted DNA/RNA may be prepared in the laboratory from an
original starting material (e.g.serial dilution of a highly concentrated tissue-
derived extract) by spiking a suitable environmental matrix. The matrix into
which the analyte is placed or diluted should be identical to, or resemble as
closely as possible, the samples that ultimately will be tested in the assay.
Wherever possible, large quantities of these reference samples should be
collected or prepared and preserved for long-term use.
4 Check analytical specicity
Analytical specicity is the ability of the assay to distinguish the target
analyte (i.e.the nucleic acid sequence of the target species) from non-target
analytes (OIE 2021). The assessment is qualitative, and the choice and
sources of sample types, organisms and sequences for evaluation of
analytical specicity should reect test purpose and assay type.
Specicity tests should assess cross-reactivity of the assay against
co-occurring species and species that are closely related to the target
organism.

Environmental DNA test validation guidelines 17
5 Check analytical sensitivity
The limit of detection (LOD) is a measure of the analytical sensitivity of an
assay. The LOD is the estimated amount of analyte in a specied matrix
that would produce a positive result at least a specied percentage of the
time. The LOD is based on detection/non-detection criteria and describes
an assay’s ability to detect the target sequence at low levels (Klymus
etal. 2020b).
The LOD should be assessed using 10-fold dilutions of the target analyte in
a suitable matrix. Dilutions can be completed using dierent matrices and
reference samples to compare the LOD of the assay between tissue-derived
and environmental DNA; however, the LOD of the eDNA assay should be
ultimately estimated using suitable environmental reference samples.
The LOD of an eDNA assay should be assessed as the last dilution showing
100% positive amplication across all technical replicates. A more accurate
estimate may be obtained by a second-stage experiment using narrower
intervals in the dilution scheme, focusing on the region between 100%
and 0%.
6 Check repeatability
Repeatability is the level of agreement between assay results, which can be
quantied within and between relevant hierarchical levels, such as machine
runs and operators, when applying the same test method within the same
laboratory (OIE 2021). Repeatability can be used to dene the expected
precision of an assay in detecting a range of analyte concentrations under
normal operating conditions.
Repeatability is estimated by evaluating variation in results from a minimum
of 3conrmed eDNA-positive independent samples (samples collected
during dierent sampling events or at dierent locations, using cleaned or
sterile equipment) within the operating range of the assay – samples should
range from well-detectable concentrations of target DNA to concentrations
close to the LOD. Initial tests for repeatability should assess assay sensitivity
with DNA reference samples using 6technical replicates in at least
5separate runs completed on multiple days. The variation in results should
be explored within technical replicates of each sample within and between
runs, expressed as standard deviations or coecients of variation (standard
deviation ÷ mean of replicates).

Environmental DNA test validation guidelines 18
For control samples to provide valid inferences about assay precision, they
should be treated in exactly the same way as test samples in each run of
the assay, including the use of blank controls, extraction-negative controls,
positive controls and standard dilutions to assess analyte concentration. It
is not acceptable to prepare a nal working dilution of a sample in a single
tube from which diluted aliquots are pipetted into reaction vessels, or to
create replicates from one extraction of nucleic acid rather than preparing
new dilution standards for each run. Such ‘samples’ do not constitute valid
replicates for repeatability studies (OIE 2021).
7 Check reproducibility
Reproducibility is the ability of a test method to provide consistent results, as
determined by estimates of precision, when applied to aliquots of the same
samples (OIE 2021). Assay reproducibility is required for assay recognition
and implementation where assays have been designed for the purposes
of biosecurity and diagnostics – these assays should be tested in dierent
laboratories using the identical assay (protocol, reagents and controls). For
routine surveillance outside the scope of biosecurity, assays can be tested
for reproducibility by individual laboratories, although inter-laboratory
testing is highly recommended.
To assess the reproducibility of an assay, each of at least 3laboratories
should test the same panel of samples (blinded) containing a suggested
minimum of 20samples, with identical aliquots going to each laboratory.
Measurements of precision can be estimated for both the reproducibility and
repeatability data.
The reproducibility process should be designed to suit the intended
purpose of the assay. For example, if the results are intended to be used
in biosecurity compliance applications, more laboratories should be used
and/or more samples should be tested to increase condence in the
results. If tests are designed to be used by a single laboratory, establishing
repeatability across operators, relevant equipment, batches of consumables
and time may be sucient.

Environmental DNA test validation guidelines 19
8 Determine thresholds (cut-os)
Selection of the assay cut-o values should reect the intended purpose
of the assay and its application. Options and descriptive methods for the
best way to determine the cut-o values of eDNA qPCR assays are available
(Caraguel etal. 2011). The main diculty in establishing assay cut-o
values based on environmental reference samples is the lack of diverse
environmental matrices that are representative of the target species and its
habitat. Moreover, eDNA extracts may contain ampliable traces of target
DNA that fall outside the dened cut-o values of the assay. Despite the
utility of cut-o values in molecular assays, any amplication considered
positive, regardless of whether it falls within or outside assay cut-o values,
must be conrmed by sequencing before conrming results.
Summary of key steps in species-specic
qPCR assay development and validation
Step 1 Attheoutsetoftheproject,denetheintended
purposeoftheassay
Step 2 Designandtesttheassay:
• Identify whether an assay exists for the intended purpose.
• Identify whether an assay exists for similar purposes or
species that could be adapted for the intended purpose.
• Identify environmental factors that may aect DNA integrity
and detection success (e.g.temperature, moisture, bacterial
activity, UV radiation).
• Identify natural/human-made inhibitors that may aect
detection success (e.g.algae, humic substances, suspended
sediment, chemical contaminants).
• Design probes and primers to target short DNA fragments
(80–280base pairs) following currently accepted barcode
regions, if no suitable assay exists.
• Test the specicity of the designed or identied assays in silico
using sequence tools against online repositories.
• Test the robustness of the designed or identied assays in
inhibition testing.

Environmental DNA test validation guidelines 20
Step 3 Validateandoptimisetheassay:
• Collect reference samples containing both the analyte and the
environmental matrix.
- Collect a sample that contains the target species and analyte
(e.g.sera, uids, tissues, excreta).
- If no known sample exists, make a reference sample by
creating a matrix that resembles the natural environmental
setting of the target species, and spiking it with the analyte.
• Test assay robustness using known concentrations of reference
eDNA samples spiked into representative environmental
matrices.
• Test the assay sensitivity using serial dilutions of reference
samples to determine the level of analyte that can be successfully
detected.
Step 4 Checkanalyticalspecicity:
• Assess cross-reactivity of the assay against co-occurring species
and species that are closely related to the target organism.
Step 5 Checkanalyticalsensitivity:
• Assess the LOD using 10-fold dilutions of the target analyte in a
suitable matrix.
Step 6 Checkrepeatability:
• Evaluate variation in results of independent replicates from a
minimum of 3conrmed eDNA-positive samples within the
operating range of the assay.
Step 7 Checkreproducibility:
• Arrange for at least 3laboratories to test the same panel of
samples (blinded) containing at least 20samples, with identical
aliquots going to each laboratory.
Step 8 Determinethresholds(cut-os):
• Use your preferred method to determine thresholds, with
the understanding that amplication must be conrmed by
sequencing.

Environmental DNA test validation guidelines 21
Resources
• eDNA Validation Scale: https://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/e dn3.189.
• Furlan EM &Gleeson D (2016). Improving reliability in environmental
DNA detection surveys through enhanced quality control. Marine and
Freshwater Research68(2):388–395, doi:10.1071/MF15349.
• Furlan EM, Gleeson D, Hardy CM & Duncan RP (2016). A framework for
estimating the sensitivity of eDNA surveys. Molecular Ecology Resources
16(3):641–654, doi:10 .1111/ 17 55 - 0 9 9 8.1 24 8 3 .
• Wilkes Walburn J, Rourke ML, Furlan E, DiBattista JD, Broadhurst MK,
Fowler AM, Hughes JM & Fielder S (2022). Robust environmental DNA
assay development and validation: A case study with two vulnerable
Australian sh. Aquatic Conservation 32(7):1225–1231, doi:10.1002/aqc.3809.

Environmental DNA test validation guidelines 22
Metabarcoding assay
development and validation
Metabarcoding is the parallel sequencing of complex bulk
samples through the analysis of short, conserved gene regions.
Metabarcoding assays have the capacity to inform users about
the presence of multiple species present in multiple samples
simultaneously, providing valuable information for community-
level species assemblages.
Environmental DNA assays designed for metabarcoding and high-throughput
sequencing (HTS) can be validated and designed within experimental
set-ups. Experimental set-ups to formally assess the performance of
metabarcoding assays can comprise mesocosm studies representative of
the target environment and species. Given the complexity of metabarcoding
assays and their corresponding data analysis requirements, developing
metabarcoding assays requires validation not only of the molecular assay
itself but also of the analytical pipeline used to curate and analyse the
resulting high-throughput data.
Steps for metabarcoding assay development and validation are
1 Dene the intended purpose of the assay (dene the targeted taxa and
outline reference database)
2 Design and test the assay (develop and test the associated bioinformatic
pipeline for data analysis)
3 Validate and optimise the assay using reference samples
4 Check reproducibility.

Environmental DNA test validation guidelines 23
1 Dene the intended purpose of the assay
The purpose of the assay must be dened at the outset, to act as a
benchmark for testing and validation (see Assay purpose and selection).
The purpose should be dened in terms of:
• the high-level purpose (e.g.to detect the range of a target species in a
certain area, to contribute to managing invasive species in an area)
• the target organisms
• what aspect of the target organisms will be measured (e.g.presence of live
organisms, presence of excreta, presence of cells)
• the sampling matrix.
2 Design and test the assay
The robustness of metabarcoding assays relies heavily on the design quality
of primers and the availability of reference DNA sequence databases at
suitable resolution to address the question. Assays must be designed and
tested in accordance with the assay’s intended purpose.
Assay choice or design
The rst step is to ascertain whether assays have already been developed
for the intended purpose or are available for testing. The length of the
targeted amplicons and gene regions (i.e.barcode) must be considered when
designing PCR primers for metabarcoding. The barcode cannot be too short
because it must be taxonomically well resolved and should span sucient
genetic variation to distinguish closely related species. However, it also
cannot be too long, because it otherwise does not t technical features of
current sequencing technologies.
Currently, most of the barcodes used in metabarcoding studies range
between 200 and 500base pairs (Pawlowski etal. 2020). Shorter barcodes
(less than 120base pairs) are sometimes used, especially for microbial
species detection, but such short gene fragments persist longer in the
environment and therefore may provide information that is less well
resolved in time and space, and has lower taxonomic resolution (Pawlowski
etal. 2020). Fusion-tagged primers (i.e.primers designed to include unique
tag identiers and sequencing adapters) can simplify library preparation
steps, and improve control of tag-jumping events and chimeric sequences.

Environmental DNA test validation guidelines 24
Sequencing errors during metabarcoding workows are common during PCR
amplication (Berney etal. 2004, Aird etal. 2011) and sequencing (Yoshitake
etal. 2021). These technical errors include nucleotide substitutions and
insertions introduced by the polymerase enzyme (Eckert & Kunkel 1991,
McInerney etal. 2014, Lee etal. 2016), nucleotide substitutions induced by
the DNA damage caused by temperature cycling during PCR (Potapov &
Ong 2017), and formation of chimeras (Fonseca etal. 2012). Chimeric PCR
products are generated when small DNA fragments that did not nish the
elongation during one step are carried over in subsequent amplication
steps. The nal amplicon will be a chimeric sequence that does not exist in
any living organism and is composed of 2 or more dierent DNA fragments
that originate from 2 or more dierent organisms.
Moreover, it is important to be aware that metabarcoding primers will not
amplify all DNA equally in a sample. They are likely to favour sequences
already predominant in the environment, leading to a biased abundance
ratio between DNA from dierent species (Elbrecht & Leese 2015, Piñol etal.
2015). One of the major factors driving PCR bias is the number of primer
mismatches for each species in the community – species with fewer, or no,
mismatches will amplify more eciently during PCR (Clarke etal. 2014). Gene
copy number (e.g.the number of mitochondria per cell), dierences between
species in ease of DNA extraction, and the type and position of primer
mismatches also inuence PCR eciency and bias.
PCR biases can be minimised by using quality measures and controls that
include suitable assay optimisation, using fusion-tagged primers to minimise
PCR steps and identify chimeric sequences, using high-delity polymerase
within reactions to improve replication sensitivity, and using multiple PCR
replicates per sample. Use of unique molecular identiers when designing
fusion-tagged primers or when using two-step PCR protocols (MacConaill
etal. 2018) can also increase analytical resolution, and allow greater
condence in identifying sequencing and PCR biases (Yoshitake etal. 2021).
Assay testing and mesocosm studies
Assays must be tested using appropriate environmental samples from areas
with conrmed presence of targeted species, or from appropriate mesocosm
experimental set-ups (Zaiko etal. 2021) with known species abundances.
Alternatively, articial samples or mock communities can be designed
following protocols in Zaiko etal. (2021).

Environmental DNA test validation guidelines 25
Library preparation
Amplicons produced using metabarcoding assays must be cleaned and
pooled to create a ‘library’ for HTS technologies (i.e.an HTS library). Several
methods are available for HTS library preparation and the steps needed
to prepare amplicons for sequencing. Metabarcoding assays must clearly
outline what library preparation strategy is to be used.
The following steps must be clearly outlined:
• PCR amplication regime and replication. Describe how many PCR
technical replicates per sample and per control will be used during the
selected amplication steps. Indicate the reaction mix and temperature
regime to be completed during amplication.
• Indexing strategy. Describe the method to be used for the addition of
sample-specic identiers (e.g.1-step PCR with fusion primers, 2-step
PCR using untagged primers with sequence overhangs, 2-step PCR using
primers with tags and sequence overhangs, tagged PCR and library build
on amplicon pool; see Bohmann etal. 2021).
• Amplicon pooling for library preparation. Describe how amplicons are to
be pooled for library preparation, indicating required concentrations and
how to select suitable PCR technical replicates for pooling.
• Troubleshooting. HTS and the pooling of PCR replicates is highly complex,
and entails costs associated with preventing, detecting and eliminating
errors and biases. Each metabarcoding approach has advantages and
disadvantages that end users must understand to better troubleshoot and
analyse HTS data. This section should provide assay-suitable information
on what complications are to be expected using the selected method,
addressing issues relating to cross-contamination risk, PCR amplication
eciency, chimera formation, tag jumping and index misassignment (see
Bohmann etal. 2021).
Sequencing
HTS libraries can be sequenced using several methods, including:
• sequencing by Synthesis (Illumina)
• single-molecule real-time sequencing (Pacic Biosciences)
• Ion Torrent sequencing (ThermoFisher Scientic)
• 454 pyrosequencing technology
• oligonucleotide ligation and detection (SOLiD) sequencing (Life
Technologies)
• nanopore sequencing (Oxford Nanopore Technologies)
• small genome sequencing (GenapSys).

Environmental DNA test validation guidelines 26
These dierent methods achieve dierent read lengths and reads per
run, and have advantages and disadvantages associated with sequencing
accuracy, time per run, costs and reagents. Assays must describe which
method is selected in accordance with the purpose of the assay. Sequencing
should be undertaken as per the manufacturer’s instructions. Deviations
from the standard method must be described clearly, supported by
published research.
Data analysis
Data analysis of sequenced outputs requires standardised bioinformatic
scripts that include data quality control, sample demultiplexing and
taxonomic identication against a reliable reference database (Figure1; Zaiko
etal. 2021). Scripts should be written with enough guidance for end users to
reliably run each script and understand what each step achieves in curating
the data.
There are important aspects that bioinformatic scripts should consider and
include for nal analysis and communicating results:
• Quality control measures. Outline how data will be curated by the script,
providing an indication of how many reads are being ltered by each step
from each sample. Quality control measures should consider sequence
quality of reads, pairing of paired end reads (if applicable), tag and primer
congruence, and ltering of chimeric or singleton sequences. It is essential
that the metabarcoding assay includes an indication of the accepted
minimal quality requirements, considering the application and context of
the proposed assay.
• Sequence taxonomic identication. The taxonomic assignment of curated
sequences must be completed against suitable reference databases,
either available in online repositories or prepared by users before the
analysis. Reference databases can be generated with a series of scripts
(see Arranz etal. 2020), but should always aim to include sequences
that correspond to the targeted genomic region (e.g.16S gene region,
cytochrome oxidaseI), correspond to the appropriate targeted taxonomic
group and taxonomic level, and include sequences with suitable metadata
associated with either museum accessions or peer-reviewed publications.

Environmental DNA test validation guidelines 27
3 Validate and optimise the assay using
reference samples
Optimisation aims to evaluate and adjust the most important physical,
chemical and biological computational parameters of an assay to ensure that
the performance characteristics of the assay are best suited to the intended
application (OIE 2021). To optimise metabarcoding assays, it is important to
select reference samples that are representative of the target environment.
These may be environmental samples that contain a variety of analytes
of interest and are usually harvested from environments with known
occurrence of targeted organisms. Environmental samples may be spiked
using extracted DNA from target organisms in the environment in serial
dilutions of highly concentrated tissue-derived extracts (Coghlan etal. 2021).
Mock mesocosm experiments can also be used to collect environmental
samples containing DNA from known species assemblages and abundances
under controlled conditions (Kelly etal. 2014, Evans etal. 2015).
Quality control
Sequence taxonomic ID
Web-based output
Raw data output
Phred score ≥ 30
Primer congruence ≤ 2 bp
Tag congruence 100%
Reference database comparison
Report preparation
User analysis
Final decision (presence/absence)
Figure 1
Example of a bioinformatic pipeline for analysing metabarcoding assayresults

Environmental DNA test validation guidelines 28
4 Check reproducibility
Metabarcoding assays can be assessed for reproducibility by processing
homogenised samples using standardised DNA extraction protocols, primers
and bioinformatic analyses (Zaiko etal. 2021). In this context, metabarcoding
assays should consistently provide comparable results between independent
laboratories, although these may show variation in raw results associated
with the use of dierent amplication regimes, instruments and user-
associated error (Zaiko etal. 2021). Verifying assays through an independent
laboratory is preferred.
Summary of key steps in metabarcoding
assay development and validation
Step 1 Attheoutsetoftheproject,denetheintended
purposeoftheassay
Step 2 Designandtesttheassay:
• Identify whether an assay exists for the intended purpose.
• Identify whether an assay exists for similar purposes or
species that could be adapted for the intended purpose.
• Design primers to target longer DNA fragments (200–500base
pairs) following currently accepted barcode regions.
• Address potential PCR biases (e.g.nucleotide substitutions and
insertions) during analyses.
• Test the assays using environmental samples from areas with
conrmed presence of targeted species, a mock community or
mesocosm experiments.
• Outline a library preparation strategy and create a library
forHTS.
• Sequence the HTS library.
• Filter and curate sequenced data before taxonomic
assignment, and use a curated reference database suitable
for the targeted gene region using curated sequences from
available molecular repositories.

Environmental DNA test validation guidelines 29
Step 3 Validateandoptimisetheassay:
• Evaluate and adjust the physical, chemical and biological
parameters of the assay to suit the intended application, using
reference samples representative of the target environment.
Step 4 Checkreproducibility:
• Process homogenised samples using standardised DNA
extraction protocols, primers and bioinformatic analyses.
Resources
• Arranz V, Pearman WS, Aguirre JD & Liggins L (2020). MARES, a replicable
pipeline and curated reference database for marine eukaryote
metabarcoding. Scientic Data 7:209, doi:10.1038/s41597-020-0549-9.
• 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 and plant communities. Molecular Ecology
26(21):5872–5895, doi:10.1111/mec.14 3 5 0.
• Zaiko A, Greeneld 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 & Pochon X (2021). Towards
reproducible metabarcoding data: lessons from an international
cross-laboratory experiment. Molecular Ecology Resources 22(2):519–538,
doi:10 .1111/ 17 55 - 0 9 9 8. 13 48 5 .

Environmental DNA test validation guidelines 30
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