Amplicon sequencing is a highly targeted method for analyzing genetic variations in specific genomic regions. Ultra-deep sequencing of PCR products (amplicons) can effectively identify mutations and characterize them. The general idea is to capture the target area in a targeted manner, and then perform next-generation sequencing (NGS), analyze the sequencing results, and obtain corresponding information.

Amplicon sequencing mainly includes 16S rDNA sequencing, 18S rDNA sequencing, ITS sequencing and target region amplicon sequencing. The sequence of a high-variation region of 16S/18S/ITS determined by a second-generation high-throughput sequencing platform to reflect the differences between species in the classification of bacteria, fungi, and archaea for the study of oceans, soils, and intestines, and the composition of microorganisms in the environment such as feces has an important guiding role, and is also a widely used method in phylogeny and taxonomic studies, especially in different metagenomic samples.

The overall route of amplicon sequencing



The principle of amplicon sequencing

Taking QIAGEN’s amplicon sequencing kit as an example, we briefly introduce the principle. PCR amplification is the main method of amplicon sequencing because the target DNA fragment can be enriched by PCR amplification. However, there may be errors in the PCR amplification and sequencing process that ultimately lead to false positives in the sequencing results. In order to overcome the human error introduced in PCR amplification, UMIs (unique molecular indices) are incorporated into the starting DNA material before any amplification, thereby preserving the uniqueness of the starting DNA molecule and overcoming the PCR amplification, as well as the bias introduced in the library construction process.

Each original DNA molecule carries unique UMIs. When analyzing data, the same UMIs are derived from the same DNA. All the same UMIs have the same diversity site in the DNA. If only one or several of the UMIs contain diversity sites, this diversity can be judged as a false positive. This method has good accuracy, specificity, sensitivity and versatility.

The applications of amplicon sequencing

(1) Cancer gene sequencing: Targeted cancer sequencing focuses on a set of genes, gene regions or amplicons that have known associations with cancer, pre-designed collections and custom collections, both can be used to study target-targeted genes;

(2) Disease screening: NGS can be used to simultaneously detect multiple genes to discover the pathogenic variation of genetic diseases. Compared with traditional methods, using NGS can reduce costs because traditional methods usually cost a lot and the results are uncertain, and require extensive testing;

(3) Plant and animal sequencing: Pre-set genomic region-targeted resequencing can reveal genetic variations in animals and plants that may represent beneficial mutations that can help making breeding decisions and reveal mutations associated with disease susceptibility.