Here’s what you need to know about amplicons and amplicon sequencing

The Takeaway: Advanced sequencing technologies have led the way in helping to revolutionize the genetics field over the past few years, and none has been more profound than amplicon sequencing. Amplicons are small segments of DNA that have been duplicated through a polymerase chain reaction (PCR). They have become a key tool for understanding the genetic makeup of organisms, as well as for diagnosing diseases and studying evolutionary relationships.

What are amplicons?

Amplicons are short, specific segments of DNA that have been amplified through PCR, which is a technique that makes millions of copies of a DNA sequence. These amplified segments are invaluable for genetic research because they allow scientists to focus on particular regions of an organism's genome. Amplicons are typically only a few hundred base pairs long, making them ideal for studying specific genes or regions of interest.

Why are amplicons important?

Amplicons play a crucial role in genetic applications from disease diagnosis to evolutionary studies and forensic analysis. By targeting and amplifying precise regions of DNA, researchers can use amplicons to isolate and study the genes that are responsible for diseases and also track genetic variations within populations. They can even identify individuals based on their unique genetic markers. Because amplicons make highly precise and efficient genetic analysis possible, they have become an indispensable tool in modern genetics.

What is amplicon sequencing?

Amplicon sequencing is a cutting-edge genetic technique that uses high-throughput sequencing technologies to analyze amplicons. It has become a fast favorite because it is efficient, cost-effective, and versatile.

Targeted sequencing: Instead of sequencing an entire genome, which can involve a lot of time and money, researchers can focus on specific regions of interest—targeted sequencing. This method is particularly useful when researchers are studying the genes associated with diseases, identifying specific genetic markers, or investigating genetic diversity within a population.

PCR amplification: PCR amplification creates amplicons for sequencing. In PCR, researchers repeatedly copy a specific DNA segment, which results in a ton of identical amplicons. This process is efficient, and it lets researchers generate the material they need for sequencing and analysis.

Cost-effective: Amplicon sequencing costs less than whole genome sequencing. This is because it reduces the amount of sequencing that is required. Researchers can target their efforts and resources on the specific genetic information they need and can forgo the need to sequence an entire genome.

High-throughput sequencing: Amplicon sequencing relies on high-throughput sequencing technologies that can rapidly sequence millions of DNA fragments at a time. This makes it ideal for projects with large sample sizes or multiple regions of interest.

What are the applications of amplicon sequencing?

Amplicon sequencing has a wide range of applications including:

• Disease diagnosis: Amplicon sequencing can be used to identify disease-causing mutations or variants in specific genes. This is especially valuable in cancer research, where identifying genetic mutations can help guide treatment decisions.
• Microbial ecology: Environmental scientists and microbiologists use amplicon sequencing to study the diversity of microbial communities in ecosystems such as soil, water, and the human gut. By sequencing specific marker genes, researchers can learn more about the microbial populations that are present.
• Evolutionary biology: Amplicon sequencing helps researchers understand the genetic diversity and evolution of different species. By comparing specific genes or regions across different populations or species, scientists can better understand evolutionary relationships.
• Forensic analysis: Amplicon sequencing can be used to identify individuals based on their unique DNA profiles. This is key for both criminal investigations and identifying human remains.

What are some of the challenges in amplicon sequencing?

While amplicon sequencing offers numerous advantages, there are also some challenges and considerations to be aware of.

• PCR bias: PCR amplification can introduce bias in which not all amplicons are amplified equally. This can affect the accuracy of results.
• Error rates: Amplicon sequencing is not error-free. Sequencing errors can occur during the process, and it's important to separate these errors from actual genetic variations when analyzing the data.
• Primer design: Primers must be carefully selected to make sure they focus on the target region and not another region.

What is the future of amplicon sequencing?

As sequencing technologies continue to advance, the cost of sequencing will likely continue to fall. As a result, the technology will become even more accessible, causing it to likely decrease in price further.

This will also help broaden the applications of amplicon sequencing, allowing the technology to expand into fields like personalized medicine, where genetic information can guide individualized treatment plans. Additionally, as environmental concerns and consciousness grow, microbial ecology studies using amplicon sequencing will become increasingly important to help us understand and preserve our planet's ecosystems.

What role does IDT play in amplicon sequencing?

IDT’s xGen™ NGS Amplicon Sequencing solutions cover both predesigned amplicon panels and custom amplicon panels, with experts on hand to help customers through the design process. IDT also offers the xGen NGS Solutions Builder Tool that helps make selecting the correct solutions a breeze.

xGen Amplicon Sequencing technology uses multiple overlapping amplicons in a single tube, featuring a rapid, two-hour workflow to prepare ready-to-sequence libraries for research studies. The PCR1+PCR2 workflow generates robust libraries even from low input quantities of DNA that may be subsequently quantified and normalized with conventional methods or normalized using the included xGen Normalase™ reagents.

To learn even more, you can download IDT’s Targeted sequencing guide here.