What does STREAM stand for?
Sequencing The Rivers for Environmental Assessment And Monitoring.
This project aims to validate DNA metabarcoding as the mainstream approach to be routinely implemented by Environment and Climate Change Canada and Living Lakes Canada for generating biodiversity data for freshwater benthic macroinvertebrates.
Who are the main partner organizations involved in the STREAM project?
The STREAM project is a collaboration between Living Lakes Canada, Environment and Climate Change Canada, and the Hajibabaei lab in Centre for Biodiversity Genomics (University of Guelph). The project is funded by Illumina and Environment and Climate Change Canada.
What is DNA metabarcoding and how it is being used in the STREAM project?
DNA metabarcoding is used to characterize the DNA of benthic macroinvertebrates collected from a bulk sample in order to identify taxa present. Samples are blended together and DNA is extracted from the collective biomass, amplified, and compared to a known library to identify which taxa of organisms are present. Click here to view an infographic of DNA metabarcoding.
Is special sampling equipment required to collect DNA samples?
No. Benthic macroinvertebrate samples are collected according to STREAM’s Field Manual, which has been developed based on Canadian Aquatic Biomonitoring Network (CABIN) protocol. Modifications to minimize DNA contamination and preserve the sample are outlined in the STREAM Procedure for collecting benthic macroinvertebrate DNA samples in wadeable streams.
Has DNA metabarcoding using benthic macroinvertebrates been tested or verified?
Yes. DNA metabarcoding has been widely used and verified prior to this project. Please see Publications page of the STREAM website for examples. This project is investigating the potential for DNA metabarcoding application in routine biological monitoring and assessment, with participants ranging from scientists to community groups.
How is DNA metabarcoding different from other eDNA projects?
With DNA metabarcoding, actual samples of organisms are collected while eDNA usually looks at DNA traces present in sampled water, sediment, soil or feces.
For the STREAM project, benthic invertebrate samples are collected, pooled and homogenized to generate a slurry from which DNA is extracted. A small, well-characterized genetic region, known as a “barcode”, is amplified from this DNA and sequenced for comparison against a library of catalogued barcodes. This comparison is used to identify the taxa present in the sample.
Targeted eDNA analysis, by contrast, is a species-specific method used to screen environmental materials, such as water or sediment, for the presence of DNA originating from specific organisms. Given the targeted nature of the analysis, it is best suited for the detection of species at risk or invasive species, rather than biodiversity assessment.
What is the benefit of biomonitoring?
Biomonitoring is an effective tool to measure environmental health because it evaluates the condition and composition of living organisms in a given ecosystem. It is based on the idea that living organisms are sensitive to change or environmental stress and, ultimately, indicators of environmental health. While biological indicators are able to provide a signal of environmental stress, detailed investigations are required to determine the cause of the stress.
What are the benefits of using benthic macroinvertebrates in biomonitoring?
The use of benthic macroinvertebrates as indicators of aquatic ecosystem health is advantageous because:
- They reflect local impacts due to their tendency to stay in a small area, experiencing constant exposure from local pollutants.
- They reflect cumulative effects due to their relatively long lifespan (1-3 years)
- They are universal
- They are ecologically significant
- They are well-characterized/well-studied
How can I get involved in the STREAM project?
As STREAM is an ongoing project, we are looking for community groups and individuals to undergo STREAM training, certification, and sample collection.
If you are interested in joining the STREAM project, please send an email outlining your name, location, desired involvement and timeline to Tamanna at the Hajibabaei lab: firstname.lastname@example.org and Raegan at Living Lakes Canada: email@example.com.
What equipment do I need for STREAM training?
Participants are expected to bring waders, pencils, clipboards and appropriate outerwear for a single-day field course.
How does my organization benefit from participating in the STREAM project?
The STREAM project offers support to participating organizations including equipment allocation, field training, sample collection and shipping. Samples contributed to the project will be analyzed at the University of Guelph at no cost. Your organization will also receive a benthic macroinvertebrate data report from the University of Guelph for the samples collected.
By collecting benthic macroinvertebrate samples, your organization or group contributes to closing Canada’s watershed data gaps and the better understanding of aquatic ecosystem health across the country.
When is sampling conducted, and how much sampling should be done?
Benthic macroinvertebrate samples can be collected for DNA analysis any time of the year, dependent on the safe access and wadeability of streams. This is different from CABIN protocol where sampling is focused on late summer–early fall.
The collection of three distinct biological replicate samples is recommended at each site. Click here to view an infographic with biological replicates. Replicate samples are collected by sampling in a riffle (3 minute kick-net) and then repeating this two more times within the same riffle, with each sample collected upstream of the last. These samples are used for quality assurance and quality control purposes and provide more statistical flexibility for data analysis and interpretation. Refer to our replicate guide on the STREAM website for more information.
Ensure replicate samples are labeled properly, using the STREAM label format, site name followed by A, B or C.
How are benthic macroinvertebrate DNA samples preserved?
Benthic macroinvertebrate DNA samples should be preserved using propylene glycol-based antifreeze or ethanol at a concentration greater than 90%.
Acceptable antifreeze types: propylene glycol-based ONLY (no propylene glycol-ethanol mixes). The recommended brand is the Absolute Zero Waterline Antifreeze available here.
Acceptable ethanol type: ethanol anhydrous, which can be purchased over-the-counter at drug stores.
DO NOT USE FORMALIN to preserve a benthic macroinvertebrate DNA sample.
How do I decontaminate equipment?
Please see the STREAM Procedure for collecting benthic macroinvertebrate DNA samples in wadeable streams for required equipment and instructions on how to properly decontaminate sampling equipment.
Why is decontamination of sample equipment so important?
Benthic sampling and processing equipment should be decontaminated between each use to prevent the transfer of DNA-containing material between samples.
How do I properly label sample jars?
Each sample jar requires key information to ensure it is properly identified. Both the container and lid require labeling. Key information may be transcribed directly on the jar, on a piece of masking tape or on a STREAM label. Include the following information with a permanent marker:
Sampling date (MM/DD/YYYY)
Code of site
Sample jar number (e.g. 1 of 3, 2 of 3, 3 of 3)
Replicate Letter IF replicates were taken (A, B, C)
Please refer to the STREAM Shipping SOP for further detail.
How are samples handled in the field and during shipping?
The proper handing and shipping of benthic macroinvertebrate samples is outlined in the STREAM Shipping Standard Operation Procedure (SOP). Please refer to this document for details.
Prior to shipping, notify the University of Guelph (Michael Wright, firstname.lastname@example.org) by filling out both a hard and electronic copy of the STREAM Sample Manifest with the required sample information.
Samples must be shipped by a GROUND courier (such as Purolator) following Transportation of Dangerous Goods (TDG) requirements.
The TDG Act and Regulations are designed to promote public safety when goods are handled and/or transported by road, rail, air, or water. The denatured alcohol used to preserve benthic macroinvertebrate DNA samples falls under the TDG Act and Regulations and has special requirement for handling and shipping. Please make yourself aware of the requirements for handling and shipping according to the TDG Act and Regulations available here.
How long does it take to get results (i.e. benthic macroinvertebrate identifications) from DNA sample analysis?
STREAM benthic macroinvertebrate DNA samples are analyzed at the Hajibabaei lab at the University of Guelph. Sample analysis can take approximately 2 months, however, other factors such as lab capacity may affect the timeline. Sample analysis may be delayed at times due to increased workload from seasonal biomonitoring programs.
We encourage groups to submit samples immediately after collection to reduce sample backlog and to avoid all samples needing to be processed in the fall & winter periods.
What is Next Generation Sequencing (NGS)?
Next Generation Sequencing (NGS) is part of the DNA metabarcoding process. NGS generates a profile of biodiversity from collected kick-net samples. Click here to view an infographic of Next Generation Sequencing.
What can I expect in results from the DNA sample analysis?
It is possible to identify benthic macroinvertebrates to the species level, however this is dependent on the completeness and accuracy of the DNA library. At this time, there are some DNA sequences that are not yet associated with an identified species.
To ensure accuracy of taxa classification it is also sometimes more appropriate to identify taxa at a higher taxonomic level (e.g. genus as opposed to species). Work is on-going to improve DNA library information.
What is taxonomic classification?
Taxonomic classification concerns the level of which organisms (e.g. benthic macroinvertebrates) are classified. For example, the spiny crawler mayfly species, Drunella coloradensis, would have the following classification:
Species: Drunella coloradensis
What is a DNA library?
A DNA library is a collection of DNA sequences from different organisms. These libraries often match morphologically identified specimens with their corresponding DNA sequence.
An example of a DNA library is the Barcode Of Life Datasystem (BOLD) here.
How can I analyze my STREAM DNA results?
STREAM data is storied in the CABIN Database and the STREAM Data Portal.
There are currently no analytical tools available through the CABIN Database to analyze benthic macroinvertebrate data based on DNA metabarcoding identifications. The results from DNA metabarcoding identification (taxa lists) will eventually be stored in the CABIN database under the STREAM-BERGE project. This project is examining the potential application of DNA metabarcoding in the context of biomonitoring for CABIN, including the development of analytical tools for data analysis.
At the University of Guelph, STREAM samples are analyzed using the latest DNA analysis pipelines. The STREAM Data Portal then generates information on the total number of phyla, classes, orders, families, genera and species as well as taxa tables highlighting bioindicator species (i.e. species which suggest the water quality status).
Is it possible to see a STREAM data report?
An example STREAM data report template is available for distribution. Please contact us if you would like a copy.
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