Environmental DNA: Processing

Once you have collected and filtered your eDNA sample, it is time to move into the lab to extract the DNA and analyze that sample according to your biodiversity targets (single-species or community). You can choose to do this in your lab, if you have the resources to do so, or submit your samples to a commercial facility.

Processing service providers

Whether you are running qPCR assays to detect and quantify DNA of single species, or doing metabarcoding to unravel community-wide information, the laboratory processing requires dedicated machines, staff, and infrastructure. Therefore it is often preferable to send your eDNA samples to a Service Provider that can process the samples for you. 

The eDNA primer provides an excellent overview of questions to ask your Service Provider as well as a list of Service Providers that process eDNA samples in Appendix II. 

Stage outcomes

The data products that you receive from Service Providers will depend on the objective you hope to accomplish. 

Single-species (qPCR) workflow:

• The final output of single-species assays includes the sample ID and the number of gene copies estimated per sample (e.g., per litre of water) that can be matched with metadata.

Community (metabarcoding) workflow:

• Unique DNA sequences (.fasta)

• Amplicon Sequence Variant (ASV) table (.csv)

• Taxonomy Table 

Processing steps

Even if you will outsource these steps to a Service Provider, understanding the steps involved will help you better understand the resulting data. An overview of the steps used in aquatic eDNA sample processing methods is provided here and described broadly below.

DNA extraction

You first need to extract the DNA from each preserved eDNA filter. Once extracted, this DNA is stable for a long time and can be used for either single-species analysis, whole community analysis, or a combination of both approaches. 

If you are using a Service Provider then they will do this step for you.

Polymerase Chain Reaction (PCR)

For all environmental DNA-based applications, it is important to have a basic understanding of what a Polymerase Chain Reaction (PCR) is. PCR is a laboratory technique that amplifies, or makes many copies of, specific segments of genetic material from a sample. It is a highly sensitive and accurate method used to detect tiny amounts of genetic material. Whether for qPCR or metabarcoding, PCR depends on using short fragments of DNA called primers that perfectly match DNA in your sample. Only DNA that matches these primers will be amplified by PCR. These targeted segments can be specific to a particular species (e.g. Chinook salmon) or group of organisms (e.g. Fish).

Single-species workflow

A species-specific assay approach will allow you to ‘look for’ only the DNA from that focal species, while ignoring the rest of the eDNA pool (think of it as the metal detector for that needle in a haystack). The quantitative nature of this approach also allows you to count the DNA copies in a sample in order to compare the relative abundance of target DNA across different samples.

qPCR Amplification

Quantitative PCR (qPCR) is a laboratory test used to find out how much of a specific piece of DNA is in a sample. The test uses specially designed matching pieces (called primers) that bind only to the DNA being measured, along with a fluorescent marker (called probes) that gives off light. In this application, the primers are designed so that they only match the species of interest, and will not match any other DNA in the sample. 

As the test runs, the DNA is copied many times. Each time the DNA is copied, a small amount of light is released. The more of the target DNA that was present in the sample at the start, the faster the light signal increases.

The test records the point at which the light becomes strong enough to be detected. This point is called the cycle of quantification (Cq). If the light appears earlier, it means there was more of the target DNA in the sample. If it appears later, it means there was less. Therefore the output of this method is ‘quantity of DNA present’, rather than a DNA sequence. 

Data Processing

To measure how much target DNA is in your eDNA sample, its Cq value is compared to that of other reference samples, or standards, that contain known amounts of the same DNA and are analyzed at the same time. These reference samples usually contain synthetic DNA that are designed to match the target DNA exactly.

From the relationship between DNA concentration and Cq values of the DNA standards (called a standard curve), the copies of target DNA per qPCR reaction can be estimated for each eDNA sample based on the Cq value for that sample. 

The copies of target DNA per reaction can then be converted to the amount of DNA per sample. This step is very important to ensure that each sample is standardized to the volume of sample water filtered in the field and just how much DNA, which is not always the same across samples. 

Note: It is important to remember that you need to be sure to evaluate your limits of detection and quantification for each single-species qPCR test you run (or get these numbers from your Service Provider) so you can state what range of DNA concentrations you can reasonably expect to be able to detect. 

There is also another type of PCR that is used to quantify target DNA, called droplet digital PCR (ddPCR).

Community workflow

DNA metabarcoding is used when the objective is to generate broad biodiversity data of all the species present in each sample, rather than focussing on one particular species of interest. This can provide excellent baseline biodiversity data or be used as part of a long-term monitoring effort to track changes in biodiversity through time. 

Broadly, the processing steps include:

PCR Amplification

The PCR used for DNA metabarcoding is at its core a standard PCR (as described earlier) but the primers are designed so that they match several different species rather than a single target. While it’s true that all species are genetically different, closely related species (such as fish) have sections of their genomes that are identical. Metabarcoding primers target these genetic regions. 

DNA sequencing

Once samples are tagged with their unique ID tags, all the samples can be pooled or mixed together and sequenced at once. Illumina DNA sequencers are commonly used for DNA metabarcoding applications. Afterward, a computer uses these unique labels to sort the sequences back into their original samples. 

Unlike qPCR where the output is the quantity of your target DNA sequence, with metabarcoding we are actually creating a list of all the DNA sequences present in the sample. The majority of raw metabarcoding sequence data will be provided in ‘fastq’ format, which is a standardized genetic notation. At this stage, each eDNA sample will have a unique text file containing all of the genetic sequences and quality information obtained from the sample.

Bioinformatics

Processing the raw sequence output file (.fastq) is called bioinformatics. This generates a list of all unique DNA sequences that occur for each sample. Each of these unique DNA sequences is called an Amplicon Sequence Variant (ASV). Many Service Providers will include this step as a deliverable when samples are submitted for sequencing. 

The final output will include the number of copies of each ASV that was present in each sample, and each ASV corresponding to a species detected in the survey, provided in the Taxonomy table.

Resources

Next stage

If you have (received) processed eDNA data, you are ready to move on to the Analysis stage.