eDNA Flashcards
What does the ‘e’ in eDNA stand for?
Environmental
What are 3 advantages of eDNA over other survey techniques?
- More Cost Effective
- Less Invasive
- More Efficient (for surveying aquatics)
List the 7 major steps in eDNA analyses in order of their process
- Survey Design
- Sample Collection
- Sample Filtration
- Filter Preservation
- eDNA extraction
- Analysis (qPCR)
- Interpretation
What are 5 (italicized) advantages of eDNA over conventional inventory methods? Can you state all ten?
- Non-invasive (to species and less invasive to the habitat)
- Highly accurate for species detection
- Cost-effective
- Able to detect presence of pathogens that cause diseases
- Minimizes the risk of pathogen transfer between sites
- Can be completed by staff without any extensive experience in species specific surveys
- Can be completed within less restrictive phonological stages for many target taxa
- Not as dependent on or sensitive to environmental conditions
- Can detect multiple species
- Can store samples for later analysis
What are 4 major limitations of eDNA?
- Cannot accurately quantify species abundance (only presence/not detected)
- Contamination can occur and cause a false positive
- Interpretation requires the person to have knowledge of the 3 processes that influence detection
- External sources of DNA can lead to a false detection (like equipment)
Describe eDNA production
- Rate of eDNA production and release into the habitat varies across species, their life stages, and across individuals
- Cannot distinguish amongst different life stages as source of DNA
- Density of individuals in habitat also influences rate of DNA production/detection
- eDNA methods cannot indicate abundance
Describe eDNA Transport
- Rate of transport varies
- Rate of transport is unknown for most species
- Longer the organism is present in the system, the further the eDNA will be transported
Describe eDNA degradation
- Rate of degradation influences amount of eDNA present in a sample
- Lifespan of eDNA in environment depends on several factors (pH, temperature, uv)
eDNA degrades at different rates and then isn’t detectable. What is an appropriate rule of thumb for the persistence (number of days) eDNA can persist in the water source
7-21 days
One must carefully consider their study objectives before using eDNA, why?
- eDNA cannot accurately and precisely linked to the concentration of eDNA in a habitat to species abundance
When should you survey for eDNA?
- Should coincide with the breeding season of the targe species (or other appropriate biological timing windows. E.g., Late summer breeding season and low flow periods for tailed frogs
What is one advantage and one disadvantage with increasing survey effort?
- Advantage: yields high confidence in results
- Disadvantage: increases project labour and costs
What guides the level of sampling effort needed (for traditional and eDNA methods)?
- Depends on the objectives
What 4 factors will influence the number of water samples you collect at a site?
- Study objective
- Budget
- System (lotic or lentic)
- Target species
What is the recommended number of samples (true samples and control samples) that should be taken?
- Well studied, high confidence for species detection probabilities, a single sample is sufficient
- Unknown detection probabilities, triplicate samples are recommended
- For new species or practitioner, one negative control sample per processing session or event is recommended
What is the recommended spacing for samples and where should they be taken?
- Spacing up to 50 m apart
- Should be collected at locations where target species is likely to occur
What is the recommended sample volume to be taken? When should this volume be the same across sites?
- Sample volume of 1 L per sample, with three samples per site
- Should be consistent within the study design for a single study when detection probabilities will be compared
Where should they be sampled?
- Keep it consistent across sites
- Surface water to avoid catching sediment
- Lotic samples should be collected from the surface water in the thalweg
- Multiple samples from the same feature should occur in an upstream direction to prevent contamination
When sampling multiple time periods, what pattern of sampling produces ‘strong’ results, ‘stronger’ results, and the ‘strongest’ results?
- Strong: sampling across seasons within one year
- Stronger: sampling in one season across multiple years
- Strongest: sampling across multiple seasons over multiple years
It was recommended that tissue assays be collected before the field samples are collected. However, field samples can be stored until the assay is completed. How should field samples be stored (two methods)?
- Shorter term storage: filters can be preserved and stored in molecular grade ethanol in the dark and remain viable for at least 6 months
- Samples can also be folded inward and placed in coin envelope in a bag with self-indicating silica beads until desiccated
- Once DNA is extracted from the filters by the lab, they can be stored indefinitely.
Under what stream/lake conditions should you not collect samples?
- After or during moderate or heavy rainfall, or during high flow events
Avoiding cross-contamination between samples is critically important for the prevention of false positive results. What 4 steps can be done to help avoid cross-contamination
- Do not leave sample collection gear unprotected or exposed
- Ensure collector does not enter the water upstream of collecting site in lotic environment or within 3m of collection site in lentic environments (expandable pole recommended)
- Ensure the sampling occurs before other field studies occur where personnel enter aquatic habitats
- Adhere to the “Hygiene protocols for amphibian field staff and researchers” to prevent spread of infectious diseases
Describe as completely as you can the process for collecting water samples for eDNA analyses (12 steps).
- Label the lid, bottle, with time, date, site, and staff
- Three 1 L samples (depending on size of wetland) for lentic species when detection probabilities are unknown
- Clean gloves at each independent collection site and refrain from touching anything other than collection bottles
- Fill bottle with water from surface of the feature
- Put lid on loosely and shake to rinse bottle
- Empty contents away from sampling site
- Repeat the rinse process 2 more times (TRIPLE RINSE)
- Fill sample bottle from surface of feature and securely tighten lid
- Fill a total of 3 bottles (typically within 10 m from each other)
- Put sample bottles in cooler filled with ice
- Collect water quality data using water quality meter ** Always do this after samples are collected
- Decontaminate clothing
When should filtering occur?
- Within 24 hours of sample collection to reduce likelihood of degradation of DNA
What order should samples be filtered?
- In the same order they were collected
Where should filtering occur?
- In a workspace dedicated to filtration - inside with access to power and vacuum and to power refrigeration
How should samples be preserved?
- Cellulose membrane is filtered into and this membrane is preserved in a vial filled with molecular grade ethanol or a coin-envelope that is stored in a bag with self indicating silica beads (dry preservation)
How should the samples be labelled?
- Ethanol resistant marker for vial or pen or pencil for the envelope
- Indicate project ID, site name and collection date
How should the filter paper be handled once filtration is completed (just know the underlined parts).
- Decontaminated forceps
- Never should be touched by hand
How should the final samples be sent to the lab and what should be included?
- Should be shipped to a lab as soon as possible after filtration
- Should be stored in secure, cool dark environment for shipment
What cautions are needed when interpreting negative results?
- Failure to detect organism in the environment is not saying that the species is absent from the site, it was just not detected
- Repetitive sampling over multiple years will increase confidence in inferences made regarding species presence
What cautions are needed when interpreting positive results?
- May represent a true positive, or a false positive (type 1 error)
- For larger lentic or lotic water bodies, it is possible to have uneven distribution of target taxa eDNA, leading to differing results
Seven factors can affect the reliability of eDNA results, what are these?
- eDNA from target species may have been introduced via other means (feces)
- may have been introduced by contamination during any stage of process
- environmental conditions (high flows)
- Low densities of target organism in the habitat (few individuals contributing DNA to the system)
- Large distance from source organism
- Inhibition arising from suspended materials in samples causing reduced sensitivity of the qPCR and decreasing the probability of detecting DNA when it is present
- Timing of sample collection related to species phenology
