1.4 Amplicon (targeted) sequencing

There is also another DNA sequencing-based method scientists can use, called amplicon sequencing or metabarcoding.

While metagenomics sequences literally everything from a sample, sequencing in metabarcoding targets a specific genetic marker sometimes referred as a barcode. A barcode is a region of DNA that is shared by all members of a group of organisms (for example, animals), but its sequence is slightly different between the members of a specific group (for example, species of fish) which the barcode makes possible to distinguish.

Different genetic markers are used for different organisms. For example, the 16S ribosomal RNA gene is used for bacteria, while fungi and animals are identified using the Internal Transcribed Spacer region (ITS, typically ITS1 or ITS2) and the cytochrome c oxidase subunit I gene (COI), respectively.

Before being sequenced, the barcode region is copied millions of times using a specific chemical reaction called polymerase chain reaction (PCR). Since the barcode is copied up to millions of times, scientists do not need a lot of DNA (or a sample of DNA that is necessarily very high quality) to start with.

You can see how PCR works by watching the video below.

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Transcript: Video 1 Overview of how PCR works.

NARRATOR:

Polymerase Chain Reaction, or PCR, uses repeated cycles of heating and cooling to make many copies of a specific region of DNA. First, the temperature is raised to near boiling, causing the double-stranded DNA to separate, or denature, into single strands. When the temperature is decreased, short DNA sequences known as primers bind or anneal to complementary matches on the target DNA sequence.

The primers bracket the target sequence to be copied. At a slightly higher temperature, the enzyme Taq polymerase, shown here in blue, binds to the primed sequences and adds nucleotides to extend the second strand. This completes the first cycle. In subsequent cycles, the process of denaturing, annealing, and extending are repeated to make additional DNA copies.

After three cycles, the target sequence defined by the primers begins to accumulate. After 30 cycles, as many as a billion copies of the target sequence are produced from a single starting molecule.

End transcript: Video 1 Overview of how PCR works.

Video 1 Overview of how PCR works.

Metabarcoding is cheaper and faster that metagenomics, but PCR amplification sometimes is more efficient for some species and not others: this issue, called amplification bias, makes the results of metabarcoding less reliable if we want to measure which species is more abundant in the sample. Additionally, no functional information relating to the genes of individual organisms within the sample can be obtained through metabarcoding.

Some important differences between metagenomics and metabarcoding are summarised in the table below.