Next-generation sequencing (NGS) is a powerful technology that enables rapid and comprehensive analysis of genetic variation in various samples. NGS can be used for different applications, such as diagnosing inherited diseases, identifying cancer mutations, detecting infectious agents, and studying pharmacogenomics. However, NGS also generates a large amount of data that can be challenging to interpret and store. Therefore, it is often necessary to focus on specific genes or regions of interest that are relevant to the research question or clinical scenario. This is where targeted gene sequencing comes in.

What is targeted gene sequencing (NGS)?

Targeted gene sequencing is a type of NGS that selectively amplifies and sequences a subset of genes or gene regions from a sample. This allows researchers and clinicians to obtain high-depth and accurate information on the variants present in the target regions, while reducing the cost and complexity of data analysis and storage. Targeted gene sequencing can be performed using two main methods: target enrichment and amplicon sequencing.

Target enrichment (NGS) : 

Target enrichment is a method that uses biotinylated probes to capture and isolate the target regions from the genomic DNA. The probes are designed to hybridize to the target regions, and then the probe-target complexes are pulled down by magnetic beads. The captured DNA fragments are then sequenced using NGS platforms. Target enrichment can cover large regions of the genome, ranging from 20 kb to 62 Mb, depending on the experimental design. Target enrichment is suitable for detecting single nucleotide variants (SNVs), insertions and deletions (indels), and copy number variants (CNVs).

Amplicon sequencing (NGS) :

Amplicon sequencing is a method that uses PCR to amplify the target regions from the genomic DNA. The PCR primers are designed to flank the target regions, and the resulting amplicons are sequenced using NGS platforms. Amplicon sequencing can cover small to medium regions of the genome, ranging from a few hundred base pairs to a few megabases, depending on the multiplexing level. Amplicon sequencing is suitable for detecting SNVs and indels, but not CNVs.

Why use targeted gene sequencing?

Targeted gene sequencing has several advantages over other NGS approaches, such as whole-genome sequencing (WGS) or whole-exome sequencing (WES). Some of the benefits of targeted gene sequencing are:

• It focuses on the genes or regions that are most relevant to the research or clinical question, avoiding unnecessary sequencing of non-informative regions.
• It provides higher depth of coverage and sensitivity for the target regions, enabling the detection of rare or low-frequency variants that may be missed by WGS or WES.
• It reduces the cost and time of sequencing, analysis, and storage, as it generates less data than WGS or WES.
• It offers more flexibility and customization, as researchers and clinicians can design their own panels or choose from pre-designed panels that cover specific diseases or phenotypes.

How to choose the right targeted gene panel?

Targeted gene panels are sets of genes or regions that are selected based on their known or suspected associations with a disease or phenotype. There are two types of targeted gene panels: pre-designed and custom.

Pre-designed panels

Pre-designed panels are commercially available panels that contain genes or regions that are curated from publications and expert guidance. Pre-designed panels are convenient and easy to use, as they do not require any design or validation steps. Pre-designed panels are available for various diseases or phenotypes, such as cancer, inherited disorders, cardiac conditions, and autism.

Custom panels

Custom panels are user-defined panels that contain genes or regions that are chosen by the researchers or clinicians based on their specific interests or needs. Custom panels are flexible and adaptable, as they can be tailored to include any genomic region of interest. Custom panels require design and validation steps, which can be done using online tools or services provided by NGS vendors.

Conclusion

Targeted gene sequencing is a useful tool for analyzing specific mutations in a given sample. It offers high-depth and accurate information on the variants present in the target regions, while reducing the cost and complexity of data analysis and storage. Targeted gene sequencing can be performed using target enrichment or amplicon sequencing methods, and using pre-designed or custom panels. Targeted gene sequencing can benefit various fields of research and clinical practice, such as diagnosing inherited diseases, identifying cancer mutations, detecting infectious agents, and studying pharmacogenomics.

References

(1) Targeted Gene Sequencing | Focused panels targeting key genes – Illumina. https://www.illumina.com/techniques/sequencing/dna-sequencing/targeted-resequencing/targeted-panels.html.

(2) Targeted next generation sequencing as a tool for precision medicine. https://bmcmedgenomics.biomedcentral.com/articles/10.1186/s12920-019-0527-2.

(3) Next-Generation Sequencing for Infectious Diseases Diagnostics is it Worth the Hype?- AACC. https://www.myadlm.org/cln/articles/2021/september/next-generation-sequencing-for-infectious-diseases-diagnostics-is-it-worth-the-hype.