1. eDNA for biomonitoring
PART 1: general introduction
Dr. C. Van der heyden

2. Steps to be taken… eDNA: what’s that?
eDNA sampling of aquatic environment
eDNA extraction
(q)PCR Observation of results
agarose gel electrophoresis
eDNA quantitation
Next Gen Sequencing
Questions

3. 1. eDNA: what’s that? eDNA = environmental DNA
All free DNA present in the environment
Aquatic, soil,…
Originating from bacteria, plants, animals,…

4. eDNA-based monitoring: why?
Selecting
Class
Colour code
High
Blue
Good
Green
Moderate
Yellow
Low
Orange
Bad
Red
Intrusive sampling
Sieving
Classic biotic index
Determining

5. Disadvantages of classic biotic index
Many specific sampling equipment needed (e.g. wading suits, nets, sieves,…)
Moving from one sampling site to the other needs good organisation
Sampling and sample processing are time consuming
Sampling is intrusive and disturbing the environment
Animals are caught and killed, ecosystem becomes poorer
Not all animals are caught easily
Buried/fast swimming animals aren’t caught frequently, though they might be abundantly present
Technique is expensive, mostly due to its labor intensity
After sampling, samples have to be washed, sieved, picked, determined
Specific taxonomic knowledge of the macro-invertebrates indispensable

6. eDNA-based monitoring: why?
DNA extraction
Filtration
Simple water sampling
Klasse
Kleurcode
Zeer goed
Blauw
Goed
Groen
Matig
Geel
Ontoereikend
Oranje
Slecht
Rood
Molecular based evaluation of ecological water quality
Agarose gel electroforesis
Amplification via PCR
using specific primers

7. Advantages of eDNA based monitoring
Much less specific sampling equipment needed
Only some clean sampling bottles, clean gloves and boots are needed
Sampling and sample processing are done quickly
Sampling is not intrusive and does not disturb the environment
Only water is sampled, no animals are caught
All animals, also buried ones, release DNA which can be found in the water
Technique is less expensive
Water samples are filtered, DNA extracted and PCR-processed
Specific taxonomic knowledge of the macro-invertebrates not needed as primers are species specific

8. eDNA in the environment
Biotic influences
Abiotic influences
 pH
 Oxygen consumption
eDNA
 UV
 Chlorophyll a concentration
 Temperature
Strickler et al 2015; Barnes & Turner 2015

9. Pitfalls and chalenges

10. Contamination Controls are needed: In the field During DNA extraction
During PCR
Positive controls are added
DNA from tissues from the animals tested for
Pre- en post-PCR processing performed in different locations indispensable …

11. PCR inhibition Humic acids Sediment samples vs. Water samples
Filtering is performed
Serum / PCR Buffers are used …

12. Wrong DNA sequences Occuring during: PCR Sequencing Trade off
Preventing mistakes
Maintaining information

13. DNA reference database
The databases should be
Reliable & complete
Depending on the species
Commonly used databases, e.g.:
fishbol.org
mammaliabol.org
barcodingbirds.org
Focus on complete sequences
Preferably mitochondrial DNA

14. Be critical!
Molecular expertise needed for a correct interpretation of the results
Criticism concerning the results and their interpretation is needed!
No contamination?
Correct amplicons?
All controls OK?
Positive results:
 animal IS present
 animal WAS present

15. Present and Future…

16. Present and Future… Present: eDNA + PCR: eDNA + Next Gen Sequencing:
Presence/absence of specific species can be demonstrated
eDNA + Next Gen Sequencing:
Aquatic ecosystem can be studied for macro-invertebrates, fish, amphibians,…
Future:
DNA-based water quality index/DNA-based water quality classification?
Standardised protocol?

17. COST action DNAquaNet

18. COST action DNAquaNet Working Group 1 – DNA Barcode References
led by Torbjørn Ekrem, Fedor Čiampor
WG2 – Biotic Indices & Metrics
Led by Jan Pawlowski, Maria Kahlert
WG3 – Field & Lab Protocols
Led by Kat Bruce, Emre Keskin
WG4 – Data Analysis & Storage
Led by Kessy Abarenkov, Diego Fontaneto
WG5 – Implementation Strategy & Legal Issues
Led by Patricia Mergen, Daniel Hering