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# 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.

Video 1 Overview of how PCR works.
Transcript

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.

## Table 2 Comparison of metagenomics and metabarcoding

Feature | Metagenomics (shotgun sequencing) | Metabarcoding (amplicon/targeted sequencing)
--- | --- | ---
Main target | All DNA in the sample | Specific barcode genes
Amplification of DNA? | No | Yes (PCR)
What does it tell us? | Taxonomic and functional profiles | Taxonomic composition only
DNA requirements | Both high quality and quantity | Low to moderate, fragmented DNA acceptable
Quantification | More accurate for relative genome abundance | Less accurate (PCR amplification bias)
Cost | Higher | Lower

A group of scientists wants to study the types of species present in a river to monitor pollution. They have a limited budget and need results quickly.

Should they use metagenomics or metabarcoding? Give one reason for your choice.

(A text entry box would appear here, but your browser does not support it.)

### Answer

They should use metabarcoding because it is cheaper and more feasible for large numbers of samples. Metabarcoding targets specific genes, reducing sequencing costs and making it suitable for identifying species when detailed functional information is not required.

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OpenLearn - The metagenomics revolution: an introduction
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