AmpliconSequencing

Amplicon sequencing is a targeted sequencing approach in which specific DNA regions of interest are amplified by polymerase chain reaction (PCR) and then sequenced using high-throughput technologies. The method concentrates on predefined loci rather than whole-genome data, enabling deep coverage of particular targets. Common applications include bacterial 16S rRNA gene sequencing, fungal ITS regions, viral or human disease–associated gene panels, and immune receptor repertoires such as T-cell or B-cell receptors.

Typical workflow involves designing primers around the region of interest, performing PCR with sample-specific barcodes or indexes, and pooling the amplified products into a sequencing library. Sequencing is usually conducted on platforms optimized for short reads, such as Illumina, though long-read options exist for longer amplicons. Post-sequencing steps include demultiplexing, quality filtering, and adapter trimming. Data analysis varies by target: microbial studies often cluster reads into operational taxonomic units or infer amplicon sequence variants with taxonomic classification against reference databases; clinical or research panels involve alignment to a reference genome and variant calling.

Advantages of amplicon sequencing include cost efficiency and the ability to achieve deep coverage of selected regions, which improves sensitivity for low-abundance variants. Limitations include primer bias, PCR-induced errors and chimeras, and restricted capacity to detect sequences outside the targeted regions. Robust primer design, appropriate controls, and validated bioinformatic pipelines are essential. Amplicon sequencing is widely used in microbiome and pathogen studies, targeted cancer gene panels, and immune repertoire analysis.