Next-generation sequencing (NGS) is a powerful technology that has transformed the study of DNA and RNA, enabling scientists to read vast amounts of genetic information with unprecedented speed and scale. Targeted next-generation sequencing (TNGS) is a specialized approach within NGS. TNGS focuses on examining specific, pre-selected regions of an organism’s genetic material rather than sequencing the entire genome, offering significant advantages for research and diagnostics.
The Core Concept of Targeted Sequencing
Targeted sequencing concentrates on specific areas of the genome, such as particular genes, their protein-coding regions (exons), or regulatory sequences. It zeroes in on genetic material already known or suspected to be involved in a particular biological process or disease. This focus is often achieved using customized “gene panels” that include hundreds or thousands of genes related to a specific condition or pathway.
This approach provides several benefits, including enhanced efficiency and reduced costs. Limiting the scope to relevant regions generates less data, simplifying computational analysis. TNGS allows for higher sequencing depth on regions of interest, meaning each targeted DNA segment is read multiple times. This increased depth improves the accuracy of detecting genetic variations, even rare ones, which is important in fields like cancer research where mutations can be present at low levels.
The Process Explained
The TNGS workflow involves several stages, beginning with sample preparation. DNA or RNA is extracted from a biological sample (e.g., blood, tissue, saliva). The extracted genetic material is then fragmented into smaller pieces (100-300 base pairs).
Adapter sequences are attached to the ends of these fragments. These adapters help fragments bind to the sequencing platform and serve as unique identifiers (barcodes) if multiple samples are processed together. The “targeting” step, target enrichment, then occurs. Specific DNA regions are either captured using complementary probes or amplified using polymerase chain reaction (PCR).
Hybridization capture uses biotinylated oligonucleotide probes that bind to desired DNA fragments, which are then pulled out using magnetic beads. Amplicon-based methods use primer pairs to amplify only the regions of interest.
The enriched fragments are loaded onto a sequencing instrument, where the reading of targeted DNA occurs in a massively parallel fashion. Millions of fragments are sequenced simultaneously, generating raw data. Finally, this raw data undergoes bioinformatics analysis. Computational tools process signals, align reads to a reference genome, and identify genetic variations (e.g., single nucleotide changes, insertions, deletions) within the targeted regions.
Key Applications in Medicine and Research
TNGS has a broad impact across medical and research fields. It is widely used in diagnosing inherited genetic disorders, identifying specific mutations responsible for conditions like cystic fibrosis or muscular dystrophy. By focusing on genes associated with these disorders, TNGS offers a precise and efficient diagnostic tool.
In cancer research and personalized medicine, TNGS identifies genetic changes within tumors, guiding treatment decisions. For instance, it can detect specific mutations (e.g., EGFR in non-small cell lung cancer or BRAF V600E in melanoma), enabling the selection of targeted therapies. TNGS allows for the detection of mutations present at low frequencies, which is significant for understanding tumor heterogeneity and resistance mechanisms.
Pharmacogenomics, the study of how genes affect drug response, also benefits from TNGS. Targeted panels can profile genes involved in drug metabolism and response (e.g., CYP2C19), helping predict an individual’s reaction to medications and enabling personalized drug prescriptions. This approach can reveal both common and rare genetic variants that influence drug efficacy and toxicity.
TNGS is applied in infectious disease management, identifying pathogens and antibiotic resistance genes. It can detect drug-resistant strains of Mycobacterium tuberculosis, guiding clinical treatment decisions. This method is useful for identifying microorganisms directly from clinical specimens, even those with low pathogen loads.
TNGS is also used in carrier screening to identify individuals who carry a gene for a recessive disorder without showing symptoms. This is relevant for couples planning a family, as it helps assess the risk of passing on inherited conditions (e.g., cystic fibrosis or Tay-Sachs disease) to their children. TNGS panels can screen for hundreds of genes simultaneously, providing a comprehensive assessment and improving detection rates.
Targeted Sequencing vs. Other Methods
TNGS offers distinct applications compared to other sequencing approaches, such as Whole Genome Sequencing (WGS) and Whole Exome Sequencing (WES). WGS involves sequencing the entire genome, including coding and non-coding regions, providing the most comprehensive genetic overview. WES focuses on the exome, which comprises all protein-coding regions of genes and accounts for approximately 1-2% of the human genome, yet contains about 85% of known disease-causing variants.
Targeted sequencing is often preferred when specific genes or mutations are already suspected or when higher depth of coverage is needed for particular regions. For instance, in clinical diagnostics for known genetic disorders or cancer, TNGS panels offer focused analysis with excellent sensitivity for detecting rare variants. This approach generates smaller datasets, translating to reduced computational resources and faster analysis times, making it more cost-effective for routine diagnostics and large-scale screening.
While WGS and WES provide broader discovery potential for novel genetic causes of disease, they come with higher costs and greater data analysis burdens. Therefore, TNGS is selected when the research question is narrow and precise, allowing for deeper, more accurate sequencing of specific areas of interest without the expense and complexity of analyzing the entire genome or exome.