Targeted sequencing is a method that sequences specific genomic regions of interest rather than sequencing the whole genome. By focusing on particular genes or regions, targeted sequencing offers a more efficient and cost-effective alternative to broader sequencing methods like whole genome sequencing (WGS).
Targeted sequencing is particularly effective for studying complex genomic regions and is often used in clinical and research settings for detailed genetic analysis. It is also used to perform targeted follow-up studies like targeted resequencing after whole genome sequencing to further examine findings in detail.
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Principle of Targeted Sequencing
The principle of targeted sequencing involves focusing on specific genomic regions for sequencing. This method identifies and enriches target DNA segments using specialized techniques before sequencing. By focusing only on specific regions, targeted sequencing provides higher coverage of these sections which improves the detection of genetic variants, including rare ones. This targeted approach enhances sequencing depth and data quality even in difficult samples such as degraded DNA. It also reduces the time and computational resources needed for analysis making it an efficient choice for focused studies.
The process of targeted sequencing is similar to WGS but it involves an additional step called target enrichment which enriches specific regions before the sequencing process begins. Target enrichment is done by either using specific probes to capture the specific DNA sequences or by using specific primers for targeted amplification. The enriched genomic regions are then sequenced and analyzed to obtain detailed information.
Process/Steps of Targeted Sequencing
- Sample Preparation: First, high-quality DNA or RNA is extracted from biological samples of interest. The quality of the extracted nucleic acids is important as it affects the downstream steps. The nucleic acids should be carefully handled and prepared to ensure they are free from contamination and degradation.
- Library Preparation: The isolated DNA are fragmented into smaller pieces through mechanical shearing or enzymatic digestion methods. These fragmented DNA undergo end repair and addition of sequencing adaptors which is essential for binding the fragment to the sequencing platform. This library preparation step ensures that the DNA is properly formatted for enrichment and sequencing.
- Target Enrichment: This is the main step in targeted sequencing that differentiates it from whole genome sequencing. The enrichment ensures that only specific genomic regions of interest are sequenced. Two methods used for target enrichment are hybridization capture and amplicon sequencing. Both methods ensure that only the desired sections of the genome are included in the sequencing process, maximizing coverage depth and minimizing the sequencing of unnecessary regions. In hybridization-based capture, specific probes are used to capture specific DNA regions of interest while in amplicon-based enrichment, specific primers are used to amplify and enrich the regions of interest.
- Sequencing: Once the targeted DNA is enriched, the prepared library is sequenced using NGS platforms like Illumina. The sequencing process involves reading the nucleotide sequence of each fragment in the library, generating millions of short reads in parallel. The type of sequencing and the read length can affect the depth and quality of the data obtained. The sequencing output is a vast amount of data containing the nucleotide sequences of the enriched regions. Paired-end sequencing is often used as it reads both ends of DNA fragments.
- Data Analysis: Finally, bioinformatics analysis is performed. This involves several stages, starting with the alignment of the reads to a reference genome and further analysis such as identifying structural variants and annotation of identified variants. Computational tools are used for quality control ensuring that only high-quality reads are considered for further analysis.
Methods of Target Enrichment
- Hybridization-Based Capture: This method uses biotinylated oligonucleotide probes to bind and capture specific DNA regions of interest. The captured DNA is then separated using streptavidin-coated magnetic beads. Unique molecular identifiers (UMIs) can be added during the preparation to tag individual DNA molecules. UMIs help identify and remove PCR duplicates ensuring more accurate quantification.
- Amplicon-Based Enrichment: This method relies on PCR amplification using primers that match the target DNA regions. The targeted areas are amplified to enrich their presence in the sample. Specific primers amplify the target regions through PCR which selectively increases their concentration. In this method, samples must be prepared and enriched individually through PCR before being pooled for sequencing. Amplicon sequencing is faster, less costly and involves fewer steps compared to hybridization capture.
| Characteristics | Hybridization Capture | Amplicon Sequencing |
| Method | It used biotinylated probes that bind to target regions followed by capture with streptavidin beads. | It uses specific primers to amplify regions of interest through PCR. |
| Complexity and Speed | It is more complex and time-consuming due to additional hybridization and capture process. | It is simpler and faster with fewer steps involved. |
| Sample Input | It requires a larger amount of input DNA. | It requires a lower input of starting DNA. |
| Cost | It is more expensive due to the longer probe design and more complex preparation. | It is comparatively cost-effective. |
| Coverage Uniformity | It generally provides more uniform coverage across the targeted regions. | It faces difficulty in achieving uniform target coverage, particularly with complex or poor-quality samples. |
| Mismatch Tolerance | It has a higher tolerance for mismatches due to longer probes increasing success. | It has limited tolerance for mismatches between target sequences and primers which can lead to amplification failure. |
| Amplification Errors | It minimizes the risk of PCR duplicates, improving the accuracy of alignment. | It can generate more PCR duplicates introducing biases in the data. |
| Sensitivity | It offers greater specificity and better performance even in low-quality DNA samples. | It is prone to off-target amplification and may affect data quality. |
| Application | It is used for applications requiring high accuracy and coverage. | It is ideal for studies where speed and cost are important factors. |
Advantages of Targeted Sequencing
- The main advantage of targeted sequencing is its efficiency. By only sequencing the regions of interest, the added costs and computational resources needed to process irrelevant data can be avoided.
- Targeted sequencing produces a smaller and more manageable data set which reduces the complexity of data analysis making it easier and less time-consuming to process.
- It offers quicker results compared to WGS, as it requires less time to analyze the specific genomic regions.
- It provides deep coverage of selected regions which is useful to detect rare genetic variants and contributes to more accurate variant calling.
Limitations of Targeted Sequencing
- It focuses on preselected genes or regions of interest so it has a limited scope in detecting novel genetic variations outside these targets. This limited scope can result in missing potentially important genetic information outside the targeted regions.
- It does not provide a complete picture of the entire genome.
- Designing sequencing panels for targeted regions can be complex and time-consuming. Poorly designed probes or primers can lead to uneven coverage or amplification failure.
- It can be difficult to ensure uniform coverage across all target regions especially when working with samples that have low-quality DNA or complex genomic structures.
- Amplicon-based sequencing has limitations in mismatch tolerance which can result in amplification failures.
- The use of PCR in amplicon-based methods can introduce duplicates and biases which makes accurate variant calling difficult.
Applications of Targeted Sequencing
- Targeted sequencing is often used in clinical diagnostics for rapid and accurate diagnosis of inherited genetic disorders.
- It can be used to study biomarkers and genetic variants associated with diseases and understand the underlying mechanisms of complex diseases.
- It is also valuable for studying viral genomes and monitoring infectious disease outbreaks.
- Targeted panels focusing on cancer-related genes can be used to identify mutations, study tumor biology, and develop personalized treatment plans.
- It is useful for detecting rare variants that other broader methods of sequencing can miss.
- Targeted sequencing is useful in microbial profiling to identify a broad range of microbes. It is also useful in metagenomics and microbiome studies.
References
- Bewicke-Copley, F., Kumar, E. A., Palladino, G., Korfi, K., & Wang, J. (2019). Applications and analysis of targeted genomic sequencing in cancer studies. Computational and Structural Biotechnology Journal, 17, 1348–1359. https://doi.org/10.1016/j.csbj.2019.10.004
- DeWitt, J., PhD. (n.d.). Targeted next generation sequencing (NGS) | IDT. Retrieved from https://www.idtdna.com/pages/technology/next-generation-sequencing/dna-sequencing/targeted-sequencing
- Gulilat, M., Lamb, T., Teft, W. A. et al. (2019). Targeted next generation sequencing as a tool for precision medicine. BMC Medical Genomics, 12(1). https://doi.org/10.1186/s12920-019-0527-2
- Pei, X. M., Yeung, M. H. Y., Wong, A. N. N., Tsang, H. F., Yu, A. C. S., Yim, A. K. Y., & Wong, S. C. C. (2023). Targeted sequencing approach and its clinical applications for the molecular diagnosis of human diseases. Cells, 12(3), 493. https://doi.org/10.3390/cells12030493
- Targeted Gene Sequencing | Focused panels targeting key genes. (n.d.). Retrieved from https://www.illumina.com/techniques/sequencing/dna-sequencing/targeted-resequencing/targeted-panels.html
- Targeted Resequencing | Focused investigation of key genes. (n.d.). Retrieved from https://www.illumina.com/techniques/sequencing/dna-sequencing/targeted-resequencing.html
- Targeted Sequencing | Thermo Fisher Scientific – UK. (n.d.). Retrieved from https://commerce.thermofisher.com/uk/en/home/life-science/sequencing/dna-sequencing/targeted-sequencing.html
- Targeted Sequencing Approaches for NGS | Thermo Fisher Scientific – NP. (n.d.). Retrieved from https://www.thermofisher.com/np/en/home/life-science/sequencing/sequencing-learning-center/next-generation-sequencing-information/ngs-basics/targeted-sequencing-approaches.html
- Targeted Sequencing Panel: Applications and analysis of targeted genomic sequencing in disease studies – CD Genomics. (n.d.). Retrieved from https://ngspanel.cd-genomics.com/resource/research-technologies/targeted-sequencing-panel-applications-and-analysis-of-targeted-genomic-sequencing-in-disease-studies.html
