Targeted Next-generation sequencing (TNGS), also known as panel sequencing, is a high-throughput method designed to investigate only specific, predetermined regions of the genome. This technology performs millions of sequencing reactions simultaneously, allowing researchers to gather vast amounts of genetic information quickly and economically. By intentionally limiting the scope of the analysis, TNGS achieves a precise and highly efficient method for detecting genetic variations relevant to particular diseases or research questions.
Focusing the Lens: Targeted vs. Broad Sequencing
Targeted sequencing fundamentally differs from broader approaches, such as Whole Genome Sequencing (WGS) and Whole Exome Sequencing (WES), in its scope and efficiency. WGS attempts to sequence nearly all of an organism’s three billion base pairs, while WES focuses on the approximately 20,000 protein-coding genes. TNGS sequences only a selected panel of genes, which may range from a few dozen to over a thousand, based on their established link to a specific condition, like a type of cancer or an inherited disorder.
This focused approach makes TNGS inherently more efficient, as it avoids sequencing the vast amounts of irrelevant DNA data generated by WGS or WES. By concentrating the sequencing power onto a small area, TNGS generates a significantly higher number of reads for each targeted base pair, known as high sequencing depth. This concentration allows for a more confident and sensitive detection of variants within those specific regions.
The Mechanics of Selecting DNA Regions
The precision of Targeted NGS relies on a pre-sequencing step called target enrichment, where only the DNA regions of interest are isolated from the entire genomic sample. Two main technical strategies are used to prepare the DNA library: amplicon sequencing and hybridization capture. The choice between these methods depends largely on the size and distribution of the DNA regions that need to be analyzed.
Amplicon Sequencing
Amplicon sequencing is a Polymerase Chain Reaction (PCR)-based method that uses specific primer pairs to rapidly amplify small, discrete regions of DNA. This technique has a simple and fast workflow, making it suitable for sequencing a smaller number of targets, such as cancer “hotspots” or a few genes known to cause a particular disease. Because the primers are highly specific, amplicon sequencing achieves a very high on-target rate, meaning most of the resulting sequence data is the intended target DNA.
Hybridization Capture
Hybridization capture is generally used when the target regions are larger, more numerous, or widely scattered across the genome, such as in large disease panels. This approach uses biotin-labeled, synthetic DNA or RNA probes that are mixed with the fragmented sample DNA. The probes physically bind, or hybridize, to the complementary target sequences. The resulting probe-target complexes are then isolated using magnetic beads. The non-target DNA is washed away, leaving an enriched library of the sequences of interest, which allows for more uniform coverage across the targeted regions than amplicon methods.
Essential Uses in Medicine and Research
Targeted NGS has become a standard tool in several areas of medicine and research because it offers a focused, actionable analysis.
Oncology
TNGS panels are routinely used to identify actionable mutations in tumor samples from patients with cancers like non-small cell lung cancer or melanoma. These panels profile dozens to hundreds of genes, such as EGFR, KRAS, and BRAF, helping clinicians select targeted therapies or match patients to clinical trials. By consolidating the testing for multiple biomarkers into a single assay, TNGS provides a comprehensive molecular profile that traditional single-gene tests cannot achieve.
Inherited Disease Testing
TNGS allows for the efficient screening of a patient’s DNA for known pathogenic variants associated with specific conditions. Clinicians can use a focused panel for conditions like cystic fibrosis, certain cardiovascular diseases, or hereditary cancers (BRCA1/2). This approach simplifies data interpretation and provides a faster, more cost-effective diagnostic path when a specific genetic disorder is suspected.
Infectious Disease Monitoring
TNGS is valuable in infectious disease monitoring and surveillance, especially for rapid pathogen identification and tracking resistance. It can be used to rapidly type a pathogen, like a virus or bacteria, or to identify mutations that confer resistance to antiviral or antibiotic drugs. This rapid and focused analysis aids public health efforts by offering a quick turnaround time for critical epidemiological data.
The Clinical Impact of High Sequencing Depth
The high sequencing depth achieved by TNGS has profound clinical implications, particularly for the sensitivity of variant detection. Sequencing depth refers to the number of times a particular base pair is read during the process; while WGS may aim for an average depth of 30x, TNGS panels often achieve depths of 1000x or higher in the targeted regions. This redundancy increases the confidence that a detected variation is real and not a sequencing error.
This high sensitivity is necessary for detecting very rare genetic variants, which are present in only a small fraction of the cells in a sample. In cancer care, this capability is leveraged to monitor Minimal Residual Disease (MRD), where cancer cells might be present at extremely low levels following treatment. Detecting these low-frequency alleles in a blood sample, known as a liquid biopsy, allows clinicians to identify relapse earlier than with traditional imaging. High depth also allows for the reliable detection of somatic mutations in early-stage tumors or in degraded DNA samples, such as those obtained from formalin-fixed, paraffin-embedded (FFPE) tissue.
Targeted sequencing offers practical advantages over broader methods, primarily in terms of cost and speed. Because fewer data are generated and analyzed, the computational burden is reduced, leading to a faster turnaround time for results. The lower cost associated with sequencing a smaller portion of the genome also makes TNGS panels more accessible for routine clinical use.