In molecular biology, amplicons represent DNA or RNA fragments that are the source and/or product of amplification or replication events. They can be naturally formed through gene duplication. Natural gene duplication plays a crucial role in genomic evolution. In this context, an amplicon refers to a section of chromosomal DNA that has been amplified and reinserted elsewhere in the genome. Amplicons can also be artificially generated using methods like polymerase chain reactions (PCR). In PCR experiments, an amplicon refers to the product of amplification reactions, i.e., PCR product.
PCR is the most important approach for generating amplicons of interest with advantages such as specificity, sensitivity and ability to amplify impure DNA. Coupling with techniques like gel electrophoresis, capillary electrophoresis, Sanger sequencing, next-generation sequencing (NGS), the length and DNA base composition can be determined. Analysis of amplicons is very useful in exploring genes of interest in the genome and has been applied to a wide range of fields, such as scientific research, forensics, and especially clinical diagnosis.
Amplicon sequencing is a highly targeted technique that enables researchers to analyze genetic variations in specific genomic regions using a series of oligonucleotide probes to target and capture regions of interest, followed by high-throughput sequencing. The length of amplicons ranges from 100 bp to 10 Kb. The ultra-deep amplicon sequencing allows variant detection and characterization. 16S/18S/ITS amplicon sequencing is a common application of amplicon sequencing. It is a prevalent method for microbial phylogeny and taxonomy studies. Another application, the targeted gene sequencing panels, is very useful in clinics. The panels contain a set of genes or regions that have certain or suspected associations with the disease or phenotype under study.
Figure 1. The workflow of amplicon sequencing.
Advantages of amplicon sequencing:
- Enables high-sensitive detection of targeted regions through ultra-deep sequencing
- High coverage: analyzing hundreds or thousands of amplicons in a single run
- Low cost and fast turnaround time
- Microbial cultures are not required
- Allows flexibility for a wide range of experimental designs
- Can obtain and analyze amplicons even from difficult-to-sequence areas, such as GC-rich regions
The Prospect of Amplicon Sequencing
Long-read sequencing techniques including PacBio SMRT sequencing and Oxford nanopore sequencing offer complete, uniform, nonbiased coverage spanning long amplicons. With the development and availability of long-read sequencing, amplicon sequencing is becoming more efficient, accurate, and sensitive. Long-read amplicon sequencing offers ultra-deep, contiguous amplicon sequencing for amplicons up to 10 kb, enabling the discovery of diverse variants, from SNPs to large insertions and deletions. Furthermore, PacBio circular consensus sequencing can obtain up to 99.999% single-molecule base calling accuracy. There have been many applications of long-read sequencing technology on amplicon sequencing, such as full-length 16S/18S/ITS sequencing.
- Valones MAA, et al. Principles and applications of polymerase chain reaction in medical diagnostic fields: a review. Brazilian Journal of Microbiology, 2009, 40(1): 1-11.
- Gao M, et al. Next Generation-Targeted Amplicon Sequencing (NG-TAS): an optimised protocol and computational pipeline for cost-effective profiling of circulating tumour DNA. Genome medicine, 2019, 11(1): 1.