Published on September 20, 2023

Key Points and Limitations of Amplicon Sequencing

Amplicon sequencing is a highly targeted technique that allows for deep sequencing of specific genomic regions. This method has been utilized since the advent of early Sanger sequencing, where it was used to amplify and sequence small DNA fragments. With the emergence of high-throughput next-generation sequencing (NGS) technologies, amplicon sequencing has evolved and expanded significantly.

Today, it is widely used in various fields, including microbial ecology, forensics, and clinical diagnostics. It allows for the identification and quantification of rare variants, detection of low-frequency SNPs, and analysis of complex populations, proving to be a powerful tool in the scientific community.

Key Points of Amplicon Sequencing

Amplicon sequencing is a powerful molecular biology technique used to study and analyze specific regions of DNA or RNA in a sample. Here are key points about amplicon sequencing:

Targeted Sequencing

Amplicon sequencing, as a form of targeted sequencing, offers high-resolution insight into specific regions of the genome. Its targeted nature allows for a deeper, more focused examination compared to whole-genome or whole-exome sequencing. This specificity enables researchers to concentrate their efforts on regions of particular interest, reducing cost, time and data management complexities.

PCR Amplification

The Polymerase Chain Reaction (PCR) is a fundamental process in amplicon sequencing. PCR is used to amplify the specific regions of interest in DNA or RNA, making millions to billions of copies. This technique uses primers that are designed to bind to either end of the desired sequence, facilitating its replication. The amplified product, or ‘amplicon,’ is then ready for sequencing. PCR amplification not only makes sequencing more feasible but also increases the sensitivity of detecting rare variants or low-frequency SNPs in a sample.

Library Preparation

Library preparation is a crucial step in amplicon sequencing. This involves preparing extracted DNA or RNA for sequencing by incorporating sequencing adapters and sample barcodes. The adapters ensure compatibility with the sequencing platform, while the barcodes enable multiplexing of samples. This means multiple samples can be pooled and sequenced together, which greatly enhances the efficiency and cost-effectiveness of the process. Once the library is prepared and validated, it’s ready for the sequencing run.

High-Throughput Sequencing

High-throughput sequencing, also known as next-generation sequencing (NGS), is the driving force behind the efficiency and speed of amplicon sequencing. This technology allows for the simultaneous sequencing of millions of DNA fragments, providing an unprecedented scale of data output. High-throughput sequencing has revolutionized genomics research, enabling a more comprehensive analysis of genetic material at a fraction of the time and cost required by traditional methods. With NGS, researchers can obtain a broader understanding of genomic and transcriptomic structures and dynamics. With the aid of bioinformatics, the enormous amount of data generated through high-throughput sequencing can be processed, assembled, and analyzed, providing valuable insights into the genetic basis of disease, evolution, and biodiversity.


Amplicon sequencing has a broad range of applications across diverse fields of study.

Quantitative Information

Amplicon sequencing yields quantitative data about the genetic composition of a sample. The number of reads for a particular sequence provides direct information about the frequency of that sequence in the sample. This enables accurate quantification and comparison of gene expression levels, taxonomic abundances in microbial communities, or variant allele frequencies in genetic studies.

Data Analysis

Data analysis in amplicon sequencing involves several critical steps to transform raw sequence data into meaningful, interpretable results.

Error Rates

Error rates in amplicon sequencing can originate from various stages of the process, including PCR amplification, library preparation, and the sequencing itself. These errors can introduce bias, leading to misrepresentation of the true genetic composition of a sample. Steps such as rigorous quality control checks, careful primer design, and selection of high-fidelity DNA polymerases can help minimize these errors.


Amplicon sequencing is a cost-efficient approach to genetic research. The targeted nature of this technique reduces the overall expense by focusing on specific regions of interest rather than the entire genome. This specificity reduces the requirement for extensive computational resources and data storage, subsequently lowering the total project costs. Furthermore, the ability to pool multiple samples in a single sequencing run through barcode multiplexing further enhances the cost-effectiveness of the technique. Therefore, amplicon sequencing stands as a financially viable option for many researchers, promoting more extensive utilization of genetic analysis in various fields.

Challenges and Limitations of Amplicon Sequencing

Despite the numerous advantages offered by amplicon sequencing, it is not without its challenges and limitations.

  1. Primer Design: The success of amplicon sequencing is heavily dictated by the design and quality of primer pairs used in the PCR amplification step. Poorly designed primers can lead to biased amplification, off-target amplification, or even fail to amplify the target regions at all.
  2. PCR-Related Errors: PCR-related errors can compromise the accuracy of amplicon sequencing. An example of these errors is chimeras, which occur when two different DNA fragments are erroneously joined together during amplification. Other errors can include nucleotide misincorporations, which can lead to false mutation detection.
  3. Limited Scope: Amplicon sequencing is a targeted approach, focusing on specific regions of interest within the genome. As such, it may overlook important genetic variations that occur outside of these targeted regions. This can limit the comprehensiveness of the resulting data.
  4. Error Rates: Error rates in amplicon sequencing can originate from various stages of the process, including PCR amplification, library preparation, and the sequencing itself. These errors can introduce bias, leading to misrepresentation of the true genetic composition of a sample.
  5. Data Complexity: The complexity and volume of data generated by amplicon sequencing require robust, sophisticated bioinformatics tools for interpretation. Not all laboratories have access to these tools or the expertise to use them, which can be a barrier to conducting amplicon sequencing studies.
  6. Sample Quality: The quality of the starting material can greatly impact the outcomes of amplicon sequencing. For instance, degraded or contaminated DNA may lead to poor amplification, failure to amplify target sequences, or false results.

Researchers should carefully consider these challenges and limitations when planning and interpreting their amplicon sequencing experiments and may need to combine this technique with other methods to obtain a more comprehensive view of the genetic information in their samples.

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