The pre-sequencing DNA preparatory step wherein DNA sequences are either amplified through amplicon or multiplex Polymerase Chain Reaction- Based (PCR-based) sequencing or captured through hybrid capture-based sequencing is called target enrichment. Target enrichment allows sequencing only part of a whole genome, or only the region of interest. It has many applications, especially in the clinical and industrial field such as cancer research and diagnosis, tumor profiling, molecular breeding, newborn screening, infectious disease testing and surveillance, and food safety. One of the several methods that simultaneously sequence thousands to millions of DNA molecules, such as these targeted sequences, is through next-generation sequencing (NGS).
Amplicon sequencing or multiplex PCR-based Enrichment, together with Hybrid Capture-based Enrichment, are the two most common techniques for NGS target enrichment. This method relies on PCR-amplification of the targeted regions of the genome using sequence-specific primers and probes. Compared to hybrid capture-based enrichment, amplicon sequencing is said to be more cost-effective, faster, and easier to accomplish. With these advantages, amplicon sequencing is considered a more suitable option for efficient gene panel testing, as well as in production-scale applications such as industrial genomic screening and clinical diagnostics. Also, it allows sequencing with low DNA input which is good for viral detection and its sensitivity.
As mentioned, amplicon-based target sequencing uses target-specific primers for PCR-mediated amplification, thus the enrichment and selectively sequenced genomic regions of interest. This approach is often used to interrogate well-characterized genomic regions such as identifying previously known drug-resistant mutants. Specifically, this NGS method is not only used in identifying genotypic markers if virulence and drug resistance, but this approach can also be performed in strain typing.
NGS techniques, such as amplicon sequencing, play an important role the viral infection research. Targeted NGS-based assays relying on amplicon sequencing are used in testing viral drug resistance which is an important factor in the management of some clinically significant viral infections. HIV drug resistance testing is one of the most known applications of amplicon sequencing in this area and this was first introduced given its sensitivity over Sanger sequencing, as well as its clinical relevance in low-abundance resistance mutations.
Aside from its application in the characterization of virulence factors, strain typing, and antibiotic resistance marker, NGS-based amplicon sequencing, particularly of rRNA genes, is used in identifying pathogenic bacteria and fungi. It has an advantage for cases wherein there is a high suggestion of infection, but the culture is negative, and when there is a suspected mixed infection.
Amplicon-based sequencing of targeted 16S rRNA, as mentioned, has immediate clinical application, particularly in the characterization of mixed infections. Specifically, it was used in brain abscess material, cystic fibrosis sputa, lymph node biopsy tissue, and even in mastoid abscess material.
Amplicon sequencing is also used in the detection of pathogens such as in individuals from a cohort with infantile infectious diarrhea. This process was done by comparing the gut microbial composition of patients with healthy individuals. Other diseases where amplicon-based target sequencing is used include cancer and inherited diseases.