Genetic information, encoded in DNA, serves as the instruction manual for all living organisms. Next-Generation Sequencing (NGS) enables rapid analysis of this information. Targeted NGS is a focused approach, concentrating on specific, pre-selected regions of the genome, allowing for efficient and in-depth examination.
What is Targeted Next-Generation Sequencing?
Targeted Next-Generation Sequencing (NGS) is a specialized approach that sequences only specific, pre-determined regions of an organism’s genome. Unlike whole-genome sequencing, which decodes every base pair, or whole-exome sequencing, which focuses on all protein-coding regions, targeted NGS precisely isolates and sequences only genetic segments deemed relevant to a particular study or clinical question. This selectivity is achieved by designing specific probes or primers that bind exclusively to the desired DNA sequences.
These selected regions can include particular genes, specific exons (protein-coding segments within genes), or regulatory elements that control gene activity. Target regions often involve creating “panels” or “gene sets,” which are collections of genomic areas known to be associated with certain diseases or biological pathways. For example, a cancer panel might include genes frequently mutated in tumors, while a rare disease panel could focus on genes linked to inherited conditions.
Why Target Specific DNA Regions?
Targeting specific DNA regions offers several advantages over broader sequencing methods.
Cost-Effectiveness
Sequencing only a small fraction of the genome significantly reduces the expenses associated with reagents, sequencing time, and data storage. This makes large-scale studies or routine clinical tests more economically feasible.
Higher Depth of Coverage
By concentrating sequencing reads on fewer targets, each targeted region is sequenced multiple times. This allows for better detection of rare genetic variants or low-frequency mutations within a sample.
Faster Data Analysis
Less data is generated compared to whole-genome sequencing, reducing computational resources and time for processing, aligning, and interpreting the results. This quicker turnaround can be particularly beneficial in clinical settings where timely results are important for patient care.
Where is Targeted NGS Used?
Targeted NGS has extensive applications across various fields, providing precise genetic insights:
- Clinical Diagnostics: It is used for identifying mutations associated with inherited diseases, enabling accurate diagnosis and carrier screening. Panels can detect genetic changes linked to conditions like cystic fibrosis or muscular dystrophy.
- Cancer Research and Diagnostics: Targeted NGS enables the detection of somatic mutations within tumors. This can guide personalized treatment strategies or provide prognostic information, helping oncologists select therapies tailored to a patient’s unique cancer profile.
- Infectious Disease Identification: The method facilitates rapid and accurate identification of pathogens, such as bacteria or viruses, and profiling their antibiotic resistance genes. This assists in managing outbreaks and guiding appropriate treatment.
- Pharmacogenomics: Targeted NGS helps predict an individual’s response to certain medications based on their genetic makeup, optimizing drug dosages and minimizing adverse reactions.
- Forensic Science: It is employed for human identification and kinship analysis by examining specific polymorphic markers in DNA evidence.
How Targeted NGS Works
Targeted NGS involves a series of steps to isolate and sequence specific DNA regions.
Sample Preparation
The process begins with extracting DNA from a biological sample, such as blood or tissue. This extracted DNA is then fragmented into smaller, manageable pieces suitable for sequencing.
Target Enrichment
This distinguishing step isolates specific DNA regions of interest from the vast amount of non-target DNA. This is commonly achieved through methods like hybridization capture, where biotinylated probes are designed to bind to the target DNA fragments, which are then pulled out using magnetic beads. Another method is amplicon sequencing, which uses polymerase chain reaction (PCR) to amplify only the desired DNA segments.
Sequencing
Following enrichment, the captured DNA fragments are prepared for sequencing, which involves attaching adapter sequences to their ends. These adapters enable the fragments to bind to a flow cell, a specialized surface within the sequencing instrument. The sequencing instrument then reads the captured DNA fragments in a massively parallel fashion, generating millions of short sequence reads.
Data Analysis
Finally, data analysis involves using powerful computer programs to align these short reads back to a reference genome, identify the targeted regions, and detect any genetic variations or mutations present within those specific areas.