What is amplicon sequencing, and how does it work?

Amplicon sequencing is a DNA analysis method that targets and sequences specific regions of interest within a genome. Unlike whole genome sequencing, which reads all genetic material, this approach isolates and multiplies chosen DNA segments before sequencing. The technique draws its name from these amplified fragments, amplicons, which are the products of PCR (polymerase chain reaction). The method excels at analysing genetic variation within defined genomic areas across numerous samples. This selective focus makes it ideal for projects requiring in-depth analysis of specific genes, genetic markers, or variable regions that distinguish between organisms or individuals.

Modern genomic research relies on efficient methods to analyse DNA across many samples. Amplicon sequencing represents a targeted DNA analysis approach focusing on specific genomic regions rather than entire genomes. This technique amplifies and sequences selected DNA fragments, allowing researchers to examine particular genes or genetic markers with high precision and depth.

How does the amplification work?

The amplification process begins with careful primer design. These primers are short DNA fragments that attach to specific regions flanking the target sequence. They are starting points for DNA polymerase, the enzyme that builds new DNA strands. During PCR, the reaction undergoes repeated temperature cycles. A double doubling occurs after every cycle, resulting in exponential multiplication. After 30-35 cycles, millions of copies of the target region exist in the sample, providing ample material for sequencing. The specificity of this amplification step determines the quality of the entire process. Well-designed primers ensure that only the intended regions amplify, avoiding off-target sequences that could contaminate results.

Sequencing process

• Library preparation – After amplification, the DNA fragments undergo library preparation, where sequencing adapters attach to each end. These adapters contain platform-specific binding sites and often include unique barcodes that identify different samples.
• Cluster generation – On the sequencing instrument, each DNA fragment attaches to the flow cell surface and undergoes bridge amplification, creating clusters of identical sequences.

The actual sequencing uses various chemistry approaches depending on the platform. Many modern systems employ sequencing-by-synthesis, where fluorescently labelled nucleotides are incorporated into growing DNA strands. The instrument detects these fluorescent signals, converting them into sequence data.

Data processing and analysis

Raw sequence data requires extensive computational processing before yielding biological insights. Initial quality control filters remove low-quality reads and trim adapter sequences. The cleaned reads are then aligned to reference sequences or assembled into longer contiguous segments. The aligned sequences are compared against reference genomes for variant detection to identify mutations, insertions, or deletions. Microbiome studies classify the sequences into taxonomic groups based on similarity to known organisms. Advanced bioinformatics pipelines automate these steps, though expert review remains essential for data interpretation.

Applications across research fields

Amplicon sequencing serves diverse research purposes. Medical geneticists use it to screen for disease-causing mutations in specific genes. Microbial ecologists deploy the technique to profile bacterial communities by targeting the 16s rrna gene. Conservation biologists monitor population genetics through targeted marker sequences. The method particularly shines in projects requiring high-throughput analysis of the same genomic regions across many samples. Its focused approach yields deeper coverage of target areas than whole genome methods at comparable cost, making statistical comparisons more robust.

Targeted DNA analysis approach continues to advance scientific discovery across numerous fields. Its depth, precision, and cost balance make it an essential tool in the modern genomics toolkit. As sequencing technologies evolve, amplicon methods adapt, maintaining their central role in genetic analysis.