February 17, 2021

Contributing author

Amplicon sequencing – what is it and how does it work?

Amplicon sequencing is a next generation sequencing method that stands in contrast to traditional culture methods followed by sequencing the entire genome of the resulting cell samples. The technique is already having a significant impact on research costs and speed and could lead to breakthroughs in several fields. Let’s take a look at what amplicon sequencing is and how it works, before discussing some of the practical applications of this new technology. We’ll also explain why it is a complementary tool for researchers rather than a wholesale replacement for traditional cultures.

Amplicon Sequencing – What Is It?

Amplicon sequencing is a targeted genetic analysis approach in contrast to the traditional method of sequencing an entire genome before you identify the species or mutations in your sample. Amplicon sequencing analyzes genetic variations in specific genomic regions. The deep sequencing of polymerase chain reaction or PCR products or amplicons allows for efficient identification of pathogens and determination of their key characteristics. 16S rRNA sequencing is a common amplicon sequencing method used to identify and compare bacteria in a sample.

How Does It Work?

Amplicon sequencing uses oligonucleotide probes that target the genetic regions that are of interest. The genetic sequences there are analyzed using next-generation sequencing or NGS.

Amplicon sequencing is useful for identifying rare mutations in a complex sample, such as when there are cancer cells mixed in with normal cells or when you’re identifying variants of rare inherited disorders. It is also used in the analysis of complex biological samples, where there are many different “species” of bacteria mixed in. This makes amplicon sequencing a natural choice for analyzing gut bacteria.

How Is Amplicon Sequencing Done?

We’re assuming you already have the sample to be analyzed. The next step is content selection and library prep. At this point, a web-based assay design tool is recommended. You’d also need to create research panels for the sections of the genome that are of interest. The samples will then be sequenced and the amplicon data analyzed. You’ll be able to learn what species are present as well as the variations between strains. You can also rapidly identify the presence of new viral strains and determine when someone is co-infected with two or more strains at once.

What Are the Benefits of Amplicon Sequencing?

There are plenty of benefits that amplicon sequencing has over traditional methods. One study found that amplicon sequencing results in much more accurate information about microbiomes in healthy children than culturing. Next generation sequencing, or NGS, like DNA fingerprinting and polymerase chain reaction (PCR) are widely used to investigate microbiota, whether you’re analyzing a nasal swab or fecal sample.

It is also more sensitive than traditional culture methods or TCMs. The study in question found that NGS identified three-quarters of the unique bacterial species in the sample, while traditional culture methods only identified a quarter of those identified by amplicon sequencing. On the other hand, amplicon sequencing identified every species found via traditional culture methods.

In contrast, traditional culture methods allow you to identify both species and strains, but it has a bias towards bacteria that readily reproduce in the lab. This causes it to miss roughly two thirds of the strains the patient actually harbors.

Another downside of traditional methods is the time it takes to get results. You have to give the bacteria time to reproduce. Yet simplicity and familiarity mean that traditional culture methods will remain in effect, while next generation sequencing methods loom as a potential replacement due to its greater accuracy.

Amplicon sequencing may be a secondary testing method for patients whose issues aren’t diagnosed by TCM. For example, traditional cultures tend to miss gut species with slow growth rates and narrow growth requirements. These tend to be identified by amplicon sequencing alone and often cannot be cultured using TCMs at all.

Another benefit of amplicon sequencing is its speed. You can quickly identify genetic variations in a sample due to the targeted approach. You could create libraries in less than eight hours and sequence them in 18 to 32 hours. Yet you don’t sacrifice accuracy, because the method can multiplex hundreds or even thousands of amplicons per reaction.

Furthermore, amplicon sequencing can be used to analyze difficult-to-sequence areas such as GC-rich regions. That is where many tumor suppressor genes are found. Targeted sequencing is also used to focus on amplicons that have known associations with cancer, allowing tumors to be sequenced and letting doctors know when cancer has mutated.

Amplicon sequencing allows for a wider range of experimental designs. You have more flexibility as a researcher with this type of next generation sequencing method. Amplicon sequencing’s targeted approach makes it cheaper than whole-genome sequencing, too.

Amplicon sequencing is an invaluable tool for researchers whether they are tracking pandemics or seeking to identify beneficial mutations in plant species. It is already being used to help patients, though it isn’t going to completely replace traditional methods as of yet.