DNA Barcoding

What is DNA barcoding?

At the supermarket, the cashier scans the barcode of your groceries. The widths of the black stripes on the produce are read by a scanner and converted into a number. This number is unique to each grocery item so it can be looked up in a database to determine what it is and how much it costs.

In the early 2000s, a group of scientists from the University of Guelph in Ontario, Canada, proposed to apply a similar approach to life. They thought that it should be possible to find a portion of DNA that would be unique to each species, and therefore that could be used to identify species the same way a barcode is used to identify groceries at the supermarket.

In animal cells, DNA can be found in the nucleus and in the mitochondria. Different parts of the DNA evolve at different rates. Some evolve fast to distinguish between individuals of the same species. These are the parts that are used in forensic tests to identify crime suspects. Other parts evolve so slowly that they are very similar from bacteria to humans. In between these extremes, a portion of the mitochondrial genome, the cytochrome oxidase I (COI) gene has been identified to evolve at the appropriate rate to differentiate species across most animals. This gene is now considered the “barcoding gene”, and typically about 650 base pairs (the DNA letters) are sequenced.

DNA evolves by mutations: base pairs composing the DNA sequence (A, C, T and G) are substituted by other base pairs. Chance and natural selection interplay, and after generations of interbreeding, new mutations are shared across individuals of the same species.

One of the strengths of DNA barcoding compared with morphologically-based identification methods is that because it is based on genetics, it tracks the interbreeding individuals, and therefore closely matches our modern species concept (i.e., “a group of living organisms consisting of individuals capable of interbreeding and producing fertile offsprings”). However, barcoding is not without issues, and can be misleading in some (rare) situations.

How do you obtain DNA barcodes?

Obtaining the DNA barcode from an animal involves three steps:

• DNA extraction: to separate the DNA from the other cell components and make it available for amplification;
• PCR amplification: to generate many copies of the desired DNA barcode gene fragment so it can be sequenced using the Polymerase Chain Reaction (PCR);
• Target sequencing: to determine the sequence of DNA nucleotides in the amplified genes, so that they can be analyzed and used to identify the organisms.

DNA extraction

DNA stores the information that cells use to function. Because of this critical role, DNA is protected within the cells by the double membrane of the nucleus or the mitochondrion. Furthermore, because DNA is a long and thin molecule, it is wrapped around proteins. Therefore, we first need to isolate the DNA. In other words, we need to make it accessible by breaking down the cell membranes and dissociate it from the proteins it is attached to. Some enzymes in the cells also attack and digest free DNA (the nucleases) which need to deactivated so they don’t digest our gene of interest.

To this end, a cocktail of chemical products are used to break down the cell membranes and the proteins: chaotropic salts that destabilize chemical bonds, detergents to solubilize lipids from the cell membranes and proteins, and enzymes (mostly proteinases) to denature proteins and in particular the nucleases. To separate these cell debris and the DNA, most modern DNA extraction protocols involve a silica membrane that binds DNA molecules but let the cell debris go through. To remove any other potential chemical compounds (e.g., sugars, pigments), the membrane is washed several times. Finally, the membrane is dried and the DNA is released by eluting it in a buffer with a slightly basic pH to keep the DNA molecule stable for several years when stored in a freezer.

Here, we will use a faster protocol that doesn’t generate a pure DNA solution but works very well for barcoding purposes with most organisms. It uses a combination of heat, proteinases and a chelating ligand called Chelex.

How to do it? Protocol for the Chelex Extraction

1. Sterilize your forceps and scissors by flaming them over an alcohol burner. This will remove any contaminants from your instruments.

2. Cut a small piece of tissue (about 1 mm²) from your sample and place it in a 1.5 mL tube. Larger tissue samples may inhibit your PCR reactions.

3. Add 150 µL of Chelex bead solution to your 2 mL tube.

4. Add 10 µL of Proteinase K to your tube.

5. Vortex the mixtures for 20 seconds

6. Spin your tube in the centrifuge for several seconds. Make sure the centrifuge is balanced with every tube having a similar tube opposite to it.

7. Incubate your tube at 95°C for at least 20 minutes (or at 60°C overnight)

PCR amplification

Once the DNA is isolated, we want to amplify targeted sequence (DNA barcode). The amplification process produces millions of copies of the barcode. The process of generating these copies is called PCR (Polymerase Chain Reaction). It uses a mix of chemical products and enzymes to amplify a specific region of the DNA. This cocktail needs to include:

• template: a small amount of your DNA solution that was extracted from your sample;

• primers: two short, manufactured fragments of DNA of a sequence that should closely complement the two ends (forward and reverse) of the desired gene fragment, thus attach and amplify it;

• oligonucleotides: a solution of the building blocks (“letters”) of DNA (A, C, T, G) that will be assembled to create copies of the barcode sequence;

• polymerase: this enzyme will assemble the oligonucleotides to create copies of the targeted sequence, using your DNA sample as a template;

• various chemicals that create a suitable environment for the polymerase to function properly.

The PCR consists of 3 steps (Fig 1):

• Denaturation: the PCR cocktail is heated to 95°C. At this temperature, the hydrogen bonds linking the two DNA strands are dissociated, leading to single-stranded DNA.

• Annealing: the temperature is decreased to 45-65°C. This temperature is typically too high for the DNA to become fully double-stranded but is low enough that the short primer sequences can attach specifically to the part of the DNA that matches their sequences.

• Elongation: the temperature is brought back up to 72°C, the optimal temperature at which the DNA polymerase functions. The polymerase recognizes and attaches to the short double-stranded piece of DNA formed by the DNA template and the primer, and starts to use the oligonucleotides found in the cocktail to create copies of the barcode using the template DNA from the sample.

These steps are repeated 30-40 times, and each cycle doubles the number of new copies being produced. Therefore, the number of barcodes in the solution increases exponentially through time. These heating and cooling cycles are automated and take place in the thermocycler. Typically, the amplification process takes about 2.5-3 hours.

How to do it? Protocol for PCR

Before we start, we need to figure out how much of each reagent we need to use for our PCR. Each reaction uses a total volume of 25 µL (24 µL of Master Mix and 1 µL of DNA template from your extraction).

We use a pre-made Taq Master Mix (that includes oligonucleotides, polymerase, and other chemicals) that needs to be diluted with water, and to which we also need to add the primers.

For each reaction, we need:

• 12.5 µL of Taq Master Mix

• 1 µL of each of forward and reverse primers

• 9.5 µL of water

In addition to amplifying our sample, we also want to try to amplify a tube that does not contain any DNA to make sure that our Master Mix is not contaminated[1]. So, if you have 11 extractions to amplify, you need to prepare enough Master Mix for 12 samples[2]. Use the table below as a template to determine how much of each reagent you need for your reaction (volumes are in µL):

Once you are done with the math, you need to:

1. Label a 1.5 mL tube “Master Mix”

2. Label as many 0.8 mL tubes (PCR tubes) as you have samples with the sample’s extraction number, plus one more for your negative control.

3. Add each reagent (in decreasing order of volume as it makes it easier to pipette) to the 1.5 mL tube labelled “Master Mix”.

4. Vortex the Master Mix tube for 5 seconds, and then centrifuge it briefly

5. Place the PCR tubes in a rack with ice (or in a cold bloc)

6. Dispense 24 µL of master mix into each PCR tube, and close the negative control tube as soon as you are done.

7. Add 1 µL of each extraction to the PCR tube labeled with that extraction number. Be careful to avoid contamination (change tips after each sample, close the tubes as soon as you put your template in, count to keep track of where you are etc.)

8. Transfer the PCR tubes to the thermocycler, close the lid, and run the appropriate program

Checking that the PCR worked

After amplification, a small amount of the amplified sample is run on an agarose gel to determine whether the amplification was successful. A few microliters of the amplified sample is mixed with a loading dye solution. The loading dye makes the DNA solution denser than the solution in which the gel floats, so it can sink to the bottom of the gel. The dye also colors the PCR product, so we can see how fast it moves through the gel.

After loading the sample, the gel is submitted to an electrical current that drives the DNA molecules in the gel. DNA is negatively charged and will travel towards the positively charged anode. Larger molecules move more slowly than small ones, and thus will travel a smaller distance.

To visualize the DNA, we added a chemical to the gel that binds to the DNA and makes it fluoresce under ultra-violet light. If the PCR was successful and DNA was amplified, it will appear as a single band on the gel. Because DNA fragments of different sizes will move across the gel at different speeds, we can also use this process to check that we amplified the correct fragment, by seeing whether it has the expected length. The approximate length can be inferred against a “DNA ladder” (solution that contains DNA fragments of known lengths) loaded into another unused well.

Notes

• If you are having issues with your amplifications, you may also want to add a positive control (a sample you know amplified correctly in the past)

• If you prepare more than 24 samples, add additional samples to compensate for the liquid that will be lost during pipetting, count 1 extra for each 24 reactions

How to do it? Protocol for running a gel

1. Pour the gel (0.8% agarose gel with GelRed added) in the rig to cover the bottom generously (~ 1/4 in deep)

2. Position the combs to create the wells into which the samples will be pipitted

3. On a piece of parafilm, use the micropipette to mix ~2 µL of PCR product and 2µL of loading dye from each PCR sample

4. Once the gel sets (20+ minutes), pull out the combs, turn the gel 90 degrees, and add TBE buffer to fully cover the gel

5. Load each well with the mix PCR product + loading dye

6. Run the gel at about 100 V for 20 min

7. Place the gel on the UV table (don’t forget to wear appropriate eye protection)

8. Take a picture of the gel

Target sequencing

The process of sequencing “reads” the series of nucleotides in the amplified DNA sequence, and converts it to human-readable letters denoting the four bases: the A, C, T and G. This is now fully automated and done in sequencing facilities.

It uses the same approach as the PCR reaction, except that the oligonucleotides used are modified to include a fluorescing dye. When they are incorporated into the sequences during the elongation process, the fluorescing dye is released and emits light. Each type of oligonucleotide (A, C, T and G) emits a different color which is picked up by the sequencer. The succession of the different colors provides a signal that is then interpreted by a computer program to reconstruct the DNA sequence.

How to do it? Demonstration using Geneious

Unfortunately, the analysis of chromatograms requires proprietary software that is relatively expensive. For this workshop, I will demonstrate how to use it.

How do you analyze barcoding data?

For this we will use SeaView.

Step 1: Aligning sequences

DNA mutations are rare and there is a lot of DNA in each cell. So, if two species share a common mutation, it is most likely because they share a common ancestor.

During the sequencing, the machine that converts the DNA molecule in letters that can be interpreted by humans has a hard time reading the beginning and the end of the molecule. Therefore, the sequences obtained do not start or end at exactly the same position.

However, to infer the phylogenetic relationships among the sequences, we need to ensure that the base pairs are aligned so we can determine which positions are the same across sequences, and which positions have changed. These changed positions will help us decide mutations that can be used to infer shared ancestry.

How to do it?

1. Start SeaView and open the alignment file provided
2. Use the space bar and backspace on your keyboard to move the sequences around so they align
3. If you have a large alignment or if you have a difficult time finding positions that are conserved enough to build your alignment, you can use one of the automated alignment method provided in SeaView:
   • Align > Alignment options, choose muscle
   • Align > Allign all

Step 2: Building trees

We are going to use the number of base pairs that differ across the sequences in our sample, and build a tree that will help us visualize these genetic distances. Individuals that belong to the same species will have identical sequences (genetic distance = 0) or very similar sequences, while individuals belonging to different species will have significative amount of divergence. It depends of the groups and of the evolutionary of the groups, but typically, individuals that diverge by more than 2-5% will be different species.

How to do it?

1. Click on Trees > Distance Methods
2. Keep the default values and click “Go”

Step 3: Interpreting trees

Here we use a basic method (Neighbor-Joining) to build trees that gives good result with DNA barcoding data if your goal is to (1) identify species limits; (2) assign sequences to species based on existing sequences. The tree obtained with DNA barcoding data and this method approximate a phylogenetic tree but is not reliable to infer relationships among species that are not very closely related.

Trees such as the one we just generated contain a lot of information, represented in a compact way.

This is what the program will output:

To understand better this figure and to facilitate its interpretation, we annotated it:

The red dots represent the internal nodes, the blue dots the terminal nodes commonly called the tips. The black lines connecting the nodes are the branches. Branch lengths represent how closely related individuals included in the tree are. For instance, in our example:

• individual 1 and 2 have identical sequences (no horizontal distance between them) and are the same species;
• individual 3 and 4 are separated by some horizontal distance and most likely represent different species.

Neighbor-Joining is a distance based method, and the branch lengths directly represent the distance (in %) between individuals. The scale bar allows you to estimate this genetic distance.

As mentioned earlier, usually distances between 2-5% can be used as a cutoff value to delineate species. Species differ in the amount of genetic variation that characterize them and this cutoff can’t always be applied bindfoldedly.

Look at the annotated tree and make sure you agree with the species being delineated.

What can you do with DNA barcodes?

In order for species to be identified from DNA, they need to be matched against a sequence coming from an identified specimen. Thus the first goal of DNA barcoding is to build a library of sequences linked to identified specimens (knowns). Once sufficiently developed, a library allows identification of other samples simply through their DNA sequences.

In the first case, the species the barcode comes from is known or can be identified unambiguously. In other words, the whole animal is available for study, typically deposited in a natural history collection. The barcode will be submitted to a public database along with information about the specimen: its species name, collection information, picture, and the location of the deposited specimen in a collection. This sequence can then be used as a reference for other applications.

In the second case, the barcode comes from a piece of the animal, an egg, a larva or another life stage that do not allow the specimen to be identified, or is taken from a specimen where the specimen was not kept. Usually, in this situation the barcode will be matched against the available public barcodes and will be used to attempt to put a species name of the animal the tissue comes from. This is one of the strengths of barcoding: it can work on small parts, even parts that are too small or indistinct to permit identification of the animal. However, this approach only works if there a reference library of the barcodes, in other words if the animal for which you only have a part has previously been identified and barcoded.

Building a DNA barcode library

Biodiversity documentation

One of the main goal of DNA barcoding is to document biodiversity. Given the huge number of species on Earth, much work remains in building barcode libraries, even for well-known groups and areas. To get an idea of the amount of DNA sequence variation within species, it is also important to obtain DNA barcodes from multiple inviduals from a variety of locations.

These barcodes are deposited in public databases (Genbank, BOLD) so they can be retrieved, compared and matched with other barcodes generated by the community.

Species delimitation

Barcodes provide an independent line of evidence that can be used to test whether morphologically variable or similar forms are the same or different species. If two populations that are genetically different do not interbreed, their DNA will not mix and this will be apparent in different DNA sequences, even when not indicated by their morphological appearance. Thus DNA sequences can be used to document species limits, and determine whether variation in color patterns, habitat preference, behavior, size, and so forth, are simply intraspecific or indicative of separate species.

Using a DNA barcode library to identify unknowns

Life stages identification

Very often larvae and juveniles look nothing like the adults. They often exhibit fewer morphological features which can make their identification particularly challenging. A tedious, impractical, expansive and often impossible solution would be to raise the larvae or the juveniles to their adult stages to see what adult they become. However, as these early life stages carry the same DNA as their adult conspecifics, if a barcode for the adult form is available, it is possible to match the barcode from the larvae to the adult form to identify them. This approach can in turn be used to describe the morphology of the larvae or to better understand their ecology (e.g., when are the larvae released? Does this correspond to a particular lunar cycle?)

Who eats whom?

Because barcodes can be obtained from small pieces of an organism, it is possible to barcode the stomach content of a predator to identify the prey it eats. This works particularly well if the prey have been recently eaten. For instance, a recent study recently showed that some species of fish which live inside coral colonies (and therefore hard to observe), have very diverse diets of smaller fishes, crabs, shrimps and other crustaceans. This study was made possible because the vast majority of the invertebrates and fishes where the study took place had already been barcoded.

Does it always work?

Barcoding works best when one (or a few) barcode matches exactly one species. This one-to-one (or many-to-one) correspondence insures that the barcode is specific to the species to be identified. However if the barcode is shared by multiple species, it won’t be possible to identify the source species exactly.

Two main factors can be responsible for having shared barcodes between species.

• The species diverged so recently that their DNA sequences have not diverged. This can be either because the DNA of a particular group evolves very slowly (e.g. in corals), or because the morphology evolves very fast – so we can distinguish species morphologically before they have evolved distinct DNA barcodes. In these cases more involved genetic methods are needed for species identification.

• The species are (or have been) inbreeding, and thus DNA from one species has passed (introgressed) into the other, preventing identification by that part of the DNA sequence. Introgression is especially common with mitochondrial DNA, like COI.