Clare Louise 
Landscape of genomic research Amplicon sequencing tool for targeted DNA analysis. This technique has revolutionized our study of specific genomic regions across various organisms, from microbes to humans. Amplicon sequencing, targeted sequencing, is a method that focuses on sequencing specific regions of interest within a genome, unlike whole-genome sequencing, which analyzes an organism’s entire genetic makeup, amplicon sequencing zeroes in on particular DNA fragments, or amplicons, that are amplified using PCR (Polymerase Chain Reaction) before sequencing.
Advantages of amplicon sequencing
Cost-effective targeted analysis
The primary advantage of amplicon sequencing is its cost-effectiveness for targeted studies. By focusing on specific genomic regions, researchers obtain relevant data without the expense of sequencing entire genomes. This approach is beneficial when studying genetic variants, gene panel’s taxonomic markers.
High sensitivity and depth of coverage
Amplicon Sequencing allows for high sensitivity in detecting genetic variations, even when present in low frequencies. The targeted nature of this technique enables researchers to achieve greater sequencing depth for regions of interest, leading to more accurate, reliable results in heterogeneous samples or when detecting rare variants.
Versatility across sample types
This technique is highly versatile and can be applied to a wide range of sample types: DNA extracted from tissues, blood, environmental samples, and even degraded or low-quality DNA sources. This flexibility amplicon sequencing is valuable in various fields, from clinical diagnostics to environmental metagenomics.
Rapid turnaround time
Compared to whole-genome sequencing, amplicon sequencing typically offers faster turnaround times. The focused approach reduces the amount of data generated and simplifies bioinformatics analysis for quicker research workflows.
Multiplexing capabilities
Amplicon sequencing can be easily multiplexed, allowing researchers to analyze multiple targets or samples simultaneously. This capability increases throughput and reduces costs per sample it is an attractive option for large-scale studies or routine testing.
Limitations of amplicon sequencing
Despite its numerous advantages, amplicon sequencing also has some limitations those researchers.
Limited scope of analysis
By design, amplicon sequencing is limited to predefined regions of interest. While this focused approach is beneficial for targeted studies, it may miss important genetic information outside the selected regions. This limitation can be particularly challenging when studying complex genetic traits or when the relevant genomic regions are unknown.
Pcr bias and artifacts
The reliance on PCR amplification introduces biases and artefacts in the sequencing results. Factors such as GC content, amplicon length, and primer efficiency affect amplification rates, potentially leading to a skewed representation of certain sequences. This bias is especially problematic in quantitative analyses, such as microbiome studies.
Difficulty in detecting structural variants
Amplicon sequencing is primarily designed to detect single nucleotide variants (SNVs), small insertions, or deletions (indels). It is less effective at identifying larger structural variants as copy number variations (CNVs) or chromosomal rearrangements, which are crucial in certain genetic studies.
While amplicon sequencing generates less data compared to whole-genome sequencing, interpreting the results still is complex. Distinguishing true genetic variants from sequencing errors or PCR artefacts requires careful analysis and often necessitates validation through other methods.
