August 2021

eDNA Frontiers Is Demystifying Earth’s
Environmental Biodiversity Through NGS

The group sequences environmental DNA to detect which organisms live in an ecosystem, assist
in biodiversity audits, identify invasive species, contribute to the conservation of rare and elusive
organisms, and more.
applications,” says Shane Herbert, business manager at eDNA frontiers.
“And there are still many new applications we’re coming across.”
iCommunity spoke with Shane Herbert about eDNA frontier’s workwith environmental DNA, its current applications in countries likeThailand and Australia, how eDNA might be used in the future, andwhy Illumina NGS technologies like MiSeq and iSeq are essential forsequencing eDNA.
Q: What is eDNA? How can it be used to measure biodiversity
and study the ecosystems around us?
SH: All living organisms leave behind deposits of themselves—
skin cells, scales, hair, feces—which hold genetic material. Theirabandoned DNA contains regions that tell scientists not only about theorganisms themselves, but also their taxonomic information. We cantarget these regions in the collected eDNA, sequence them, and thenfind a taxonomic match in global DNA databases.

Shane Herbert is the business manager at eDNA frontiers, a researchservice group out of Curtin University in Australia

Introduction
As organisms move through the world, they shed genetic material.
Cells from their fur, skin, scales, and other sources end up in theenvironment, providing genetic clues about what organisms exist
there. For a long time, this “environmental DNA” or “eDNA” went
unappreciated by researchers. Now, eDNA frontiers, a researchservice group out of Curtin University, has figured out how to wieldenvironmental DNA to help industry, government, and researchers
study biodiversity and ecosystems.
The group, based in Perth, Western Australia, is analyzing eDNA
to reveal what organisms exist in various habitats. eDNA frontiers
takes samples from the environment and then uses Illumina’s NGS
technology to sequence the DNA contained in them. The sequencing
data can identify the organisms present in the eDNA sample and thus,
which ones have been in that location.
eDNA frontiers says the technique is faster and can be morecomprehensive than scientists’ traditional methods of collecting andidentifying organisms. The applications of eDNA are also vast—theyinclude biodiversity audits, monitoring for invasive species, dietanalysis, finding rare and elusive organisms for conservation, andmore. “We’ve got clients from all around the world doing environmental
impact assessments, biodiversity studies, and many other

For Research Use Only. Not for use in diagnostic procedures.

“By doing it that way rather than using
a qPCR method, you get information
not just about one species, but all thespecies targeted by that assay. You endup with a broader list of the organisms
living in that environment and this gives
you more interpretive power.”
You pick up things you wouldn’t ordinarily be able to see, such asorganisms in larval stages or elusive organisms that hide during all
stages of their development. Virtually all of them leave DNA traces inwater or in other mediums, which makes them detectable. It’s a great
method for quick and economical audits of biodiversity.
Biodiversity audits are currently the main use of eDNA, but its
applications are growing. You could go into an environment with little
idea of what’s there and target it with a general or narrow assay. If
you’re looking more generally, you could use a targeted nuclear gene
like 18S to find all the eukaryotes, or you can use a more specific
FISH assay to narrow it down to the species level. These can be used
to guide interpretations in environmental impact statements whendisruptions to ecosystems occur. Or you can go hunting for invasive
species or rare and elusive species. It’s great for diet analysis too.

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August 2021
Q: What are the biggest challenges of working with eDNA?
SH: The typical eDNA samples we handle are filtered water, which isa water sample that we filter onto a membrane and then collect thebiological material in it. But it’s important to think about how you doyour sampling. If you’re looking for a marine species, can you pick it
up in the water? Or do you have to take a scraping to detect it? Do
you do replicates and if so, in which locations? Then of course there’sextraction. You can’t just assume your extraction method covers
everything. You need to make sure that the DNA in the cells you’vecollected can be made available for analysis. The other issue withenvironmental samples is that you’re typically dealing with degradedmaterial. The cell is in the process of breaking down, so you’re not
likely to find a whole genome—it’s going to be in little fragments.

If you happen to amplify something else that shares the same primer
or probe sequence, then you don’t have any way of knowing if thesignal is off target. Equally, if qPCR is negative then you assume it isnot present. This is where the sequence information is quite useful
because it is its own positive control.

“You can be confident a positive istrue since the target sequence isthere in the data, and if there aresequence variants that otherwise
knock out a qPCR assay, you’ll see
it and rescue a false negative. This isa major advantage with NGS where
we can see the variation within theamplicons that we sequence.”
It’s interesting in a biomonitoring study where we take samples from a large
geographical region and you see a bit of sequence variation across theregion. It can start telling you about the connectivity between those sites.
It’s not population genomics, but it’s still useful information.
Q: What is the NGS method you use to analyze biodiversity inenvironmental DNA samples? What is the metabarcoding approach?

A researcher collecting a sea water sample, the water sample isthen filtered onto a membrane and the biological material in it iscollected and processed at eDNA frontiers.

Another challenge is just how many species exist in the world. There arean enormous number of unknown organisms. We run all our samples
against GenBank as well as custom databases, and the number ofunknowns is still incredibly high. But we could still match our unknowns
in the future when a species’ DNA is finally sequenced and entered into
genetic databases. This remains a global biodiversity “to-do.”
Q: What role is NGS playing in eDNA research? What advantages
does NGS provide compared to the qPCR method or even PCR?
SH: With NGS, you get a lot of information. It’s a broader result,it’s more identifications per sample. It gives you greater interpretive
power to look at your data and find out what’s there, what’s missing,
and what patterns correlate with other sampling area data, such as
if it’s polluted or not. You can still get the presence or absence of aparticular organism, say an invasive species or animal of conservationinterest, with good sensitivity. You’re getting that information, plus
everything else. The extra information just adds to the environmental
picture of where the sample came from and that is quite interesting.
With qPCR, you use a probe binding assay. The output is a fluorescentsignal, a yes or no. All your effort has to go into the development of thatassay, to make sure your signal is going to be what you want it to be.

For Research Use Only. Not for use in diagnostic procedures.

SH: The reason we don’t do something like shotgun sequencing in anenvironmental sample is because they’re full of bacteria and unicellularorganisms. If you’re not looking at them, it’s just chewing up yoursequence yield. What you’re really interested in is hiding in the one or
two percent of the total sample. Metabarcoding allows you to screen
everything else out and just look at what you’re interested in withgreater definition.
Also, shotgun sequencing looks at the genomic, mitochondrial, andchloroplast DNA from all the organisms in the sample. That’s a lot ofDNA. It’s also mostly unknown because it comes from species thatare barely known or described. The small numbers of those thatare taxonomically identified may have had only a couple of barcode
regions sequenced and loaded to GenBank. So even with all thesequencing data, you can’t use it to identify much. Metabarcoding is
like using a special lens where you amplify only the DNA that you canmatch with in GenBank and deliberately exclude all the DNA that’sunhelpful. A whole genome could be reduced from 3.2 billion basepairs (i.e. humans) down to approximately 200 to 300 base pairs in a
metabarcode target.
Q: What NGS system is your institute is currently using? What isthe reason that you’re using this system?
SH: We’re a big fan of the MiSeq. We’ve got a couple of those. The
main reason we use it is the sequencing length. Up to 600 cycle,
2x300, that covers all the barcodes we need to work with. They’re abit longer than the other systems Illumina has that can only do 2x150,
which isn’t long enough for some of our barcodes.

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Q: What projects is eDNA frontiers currently working on?
SH: There are a couple of great examples that are pertinent to theSoutheast Asia region. Shipping and invasive species is a big one—the chance of transfer or translocation of invasive marine species is on
a lot of people’s minds. There’s been interest in invasive species for along time, so they are already taxonomically identified and sequenced.
So, there’s good context to use for an eDNA study.
In the ports where ships end up after journeys from elsewhere, eDNA
gets sampled using floating arrays which have settlement plates
on them that allow algae and crabs and whatnot to live on them.
Maybe two months or six months later they get collected, the biofoul
is scraped off, and then we test them. We first use a generic assay
in a triage system to see if an invasive species is present. If there isone, then we do another series of tests to confirm whether a specificinvasive species is there. Ultimately, an ongoing eDNA biomonitoring
study helps support the port’s resource management plan by allowing
them to deploy remedial actions when needed, rather than generically
applying them all the time.

Illumina Next-Generation Sequencing technologies like the MiSeq
and iSeq systems are essential for sequencing eDNA, and arecompatible with biomonitoring needs.

Our MiSeqs run around the clock and they’re very nice little machines.
We’ve also got an iSeq, which we bought specifically for more mobile
barcoding methods. We put it in a protective case that’s on wheels sothat we could move it around, plug it in, load up samples, and sequence
on-site. That was an interesting little experiment and the system worked
very well. We even put it on a boat—the collaboration team went out
and collected samples in the ocean and did sequencing at sea. I think
the iSeq is quite good, it’s quite a robust little system. But certainly
MiSeq is our workhorse.
Q: What are the limitations of eDNA sequencing?

Another application for eDNA is happening on oil and gas rigs in
the Thailand Sea. It’s a big project one of the professors at Curtin
University, Euan Harvey, is working with Chevron on regarding theiroffshore platforms in the Thailand Sea, which have been there for thirty
or forty years. They’re reaching the end of their life and need to be
decommissioned. The study involves taking eDNA samples off themin deep-water before they’re taken down. They’ve got thirty or forty
years’ worth of biodiversity growing on them. They’re tremendously
lively places, there’s just so much living there.
It’s a biodiversity audit to see what’s there, but it’s also an invasive
species check. The destination for some of those decommissioned
platforms could be closer to shore for recreational and fisheries
purposes. Euan has done a number of studies that show these 3D
structures actually promote fish growth more than reefs. But before
you move it to help fisheries, you want to make sure it’s got a safe setof organisms and you’re not translocating invasive species onto local
reefs. Using eDNA helps those decisions to be made, and its much
simpler & quicker than visually ID’ing them all.

SH: We don’t get individual information on a specific organism nor are
we able to identify which animal has been where. Sequence variants
are a useful tool for connectivity information of a species, but it’s not
population genomics information. We often get asked if eDNA cando those things and for the moment it cannot. It’s also not always
possible to get to species level and this is occasionally a biology
issue—two or more species share the same sequence and we can’ttell them apart. There is also the “unknowns” problem. This is always
going to be a limitation in a DNA analysis until all organisms on Earthhave a barcode sequence.
Abundance is another issue that eDNA metabarcoding struggles with.
This is related to the complex PCR the method does, which canintroduce biases in the final products. But it’s also a biology issue. For
example, if you’re looking at fish in a pond, there might be one large
fish and fifty small fish. Smaller, younger fish don’t shed as much.
Larger, older fish shed lots of cells and release eDNA at a higher rate.
So while we can sequence and count out the sequence reads, biology
influences the result and it’s not necessarily going to be accurate. We
can estimate a relative abundance, but it’s just not something we tend
to rely on. Ironically, qPCR is slightly more helpful in that respect. But
even then, it still needs to be assessed for every type of organism.

For Research Use Only. Not for use in diagnostic procedures.

eDNA is a great way of doing biodiversity audits on organisms
without having to catch and kill them such as species like theweedy seadragon which is endemic to Australian temperatemarine waters.

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August 2021
Also, in sampling these deep-water jackets, you can get DNA of rareand elusive organisms that aren’t well represented in other studies.

all over the place, but that didn’t seem to be the case so its degrading
quickly enough to allow applications like that to make sense.
Q: How do you envision eDNA being studied or used in the future?

“eDNA is a great way of targetingthese organisms without having tocatch and kill them. It gives you achance to detect them from what
they’ve left behind rather than fromthe organisms themselves.”
These organisms’ feces are also interesting because you can do aneDNA study and get a diet profile of what they’re eating, which tells
you something about the environment they live in. This plays directly
into conservation management of species.
Q: What advice would you give researchers and companies
interested in using eDNA technology?
SH: My first piece of advice is to know what you’re looking for. We cantake a sample, sequence all of it, and tell you what’s there but it’s better
if you refine the question. Are you interested in fish? Crustaceans?
We’ve got something like 130 assays that target a lot of different
things, twenty of them for fish alone.

SH: There are a number of places this field could go, such as
developing a quicker process to help make decisions at the pointof analysis. You could take samples on the side of a waterway, forinstance, and get a rapid result that informs a management strategyon the ground in real time. Abundance is something else we would liketo address. It would make eDNA analysis wonderfully helpful to know
how many of a particular species were present at a specific time. Think
if you could monitor the migration of fish along a coastline or waterway
during breeding—it would be a great way to help better conserve themduring this delicate time.
My impression is that regulatory authorities are starting torecognize the benefit of this technique and make guidelines andrecommendations to use it for certain groups of organisms andapplications. Those will be frameshift moments for eDNA and its use
will increase. In the meantime, though, there are still many aspects
that need to be resolved so that eDNA can be used reliably. We areinvolved with a few initiatives within Australia and watching global
events closely, all of which are really encouraging. I suspect in the next
year or two this will be a formal part of environmental monitoring.

Learn more about the products and systems
mentioned in this article:

It’s also worth setting expectations appropriately—eDNA doesn’t
solve all questions. There’s still work involved in the interpretation.
We typically don’t interpret results at eDNA frontiers. We’ll tell you
what’s there based on the results but its worth noting that its harder toconfirm whats not there, as it may be a genetic variant that is missedby the assay.

Illumina MiSeq:
https://www.illumina.com/miseq

Q: How far back in time can you measure for eDNA?

This article was written in collaboration with:

Illumina iSeq100:
https://www.illumina.com/iSeq

SH: The degradation rate varies greatly. It relates to temperature,pH, bacteria, inhibiters, all those things. It’s not an easy question toanswer. eDNA does persist, but it doesn’t stay forever. Generally eDNA
doesn’t stay around for longer than hours to days. Having said that,we’ve done ancient DNA work and this can go back a very long way.
Our sister academic group, the Trace and Environmental DNA (orTrEnD) lab at Curtin University is involved in a study of a Canadian lakethat looks back over two hundred thousand years. The collaboratorgroup took an ice core sample that represents this timescale and they
could determine what plant species were in it from that long ago. So
timescales can be complex.
However there was a nice study on the deep-water oil & gas jackets inThailand, where we took water at the surface, 10 meters, 20 meters,
30 meters and at the sediment. The biodiversity profiles clustered quite
nicely—the distinct environments of the water samples we took at
each point were relative to the depth. You’d think it’s water, it moves

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