Next-Generation Sequencing in the Clinic: Opportunities and Challenges

NGS is a transformative technology used extensively in clinical diagnostics and pathology research. It is increasingly integrated into routine medical practice, primarily used as a tool by pathologists, who are specialized physicians with expertise in diagnosis, and consultants to patient-facing clinical teams, to provide information for the effective treatment of the patients.

Examples of its use include: 1) tumor profiling to identify biomarkers for targeted therapy, 2) monitoring minimal residual disease after tumor treatment, 3) detecting inherited genetic disorders (in adults, prenatal and newborn screening), 4) outbreak tracking, 5) and antimicrobial resistance profiling, etc.

According to the American Association for Cancer Research, the US Food and Drug Administration issued 13 approvals – covering 9 types of cancers and 2 disorders – leveraging biomarker-guided patient selection in the first quarter of 2025 alone.

In research, NGS is a foundational tool for understanding the biology of health and disease and has many uses:

• Genomic–wide association studies help to identify genetic variants associated with complex diseases.
• Transcriptomics measures gene expression and splicing variants.
• Epigenomics assesses DNA methylation and chromatin accessibility to understand gene regulation.
• Microbiome studies characterize microbial communities and their links to health.
• Single-cell sequencing allows for the analysis of gene expression or DNA mutations at the individual cell level, critical for studying heterogeneity.

There are several clinical tests that make use of NGS:

• Identifying hereditary disease: Whole-exome sequencing and gene panels use NGS to identify pathogenic variants causing rare genetic disorders or hereditary cancer syndromes. It is also being used to find novel causes of rare, undiagnosed syndromes and developmental delays.
• Cancer genomic testing: Tumor sequencing is done using NGS to identify somatic mutations (i.e., those mutations that occur after birth). These somatic mutations can often be used to help determine the prognosis or can help in the selection of precision treatments for cancers. For example, PARP inhibitors work to selectively kill cancer cells that have mutations in certain genes like BRCA1 or BRCA2. NGS can also be used for liquid biopsies that identify circulating tumor DNA in the blood. This is a non-invasive way to monitor cancer and see how treatments are working. This can be used to figure out if there are specific mutations in non-small cell lung cancer that are actionable without having to do an actual biopsy of the lung tumor. Researchers are working on trying to use liquid biopsies as a way to detect cancer early as well, but I believe early detection applications are still undergoing trials.
• Carrier screening: NGS allows couples to undergo testing to find out if they are carriers for a large number of autosomal recessive and X-linked genetic conditions so they can determine if their future offspring would be at risk of having one of many genetic diseases, such as Tay-Sachs.
• Pharmacogenomics: NGS is used to identify genetic variants that can influence drug metabolism. These data are sometimes used to help physicians when they are selecting medications like antidepressants.
• Non-invasive prenatal screening (often called non-invasive prenatal testing, or NIPT): A blood sample is taken from a pregnant individual and the fetal DNA that is free-floating in the mom’s blood is sequenced. Using this information, clinicians can determine if the fetus has a high risk for Down syndrome, other chromosome abnormalities and even some single-gene disorders.
• Infectious disease diagnosis: NGS can be used to help detect infections and outbreaks or track the evolution of infectious organisms.

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Next-Generation Sequencing (NGS): Definition and Overview

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As a practicing pathologist with expertise in sarcoma diagnosis, I routinely utilize NGS as a vital ancillary tool. It plays a critical role in establishing an accurate diagnosis and precise tumor classification for these rare but devastating tumors. In addition, NGS provides essential prognostic and predictive information that helps guide the clinical team in tailoring treatment strategies and optimizing patient management.

One major challenge is trying to determine if a gene change is a pathogenic variant (i.e., causes disease) or if it is just a normal variation that does not cause disease (i.e., a variant of uncertain significance). This is critical because we don’t want patients having breasts or ovaries removed if they are not at high risk for cancer in these organs. NGS lacks good coverage of certain areas of the genome, so it is important for clinicians to know that the gene they are interested in has good coverage, otherwise, they may get a false negative result (i.e., the pathogenic variant may not be found).

NGS is not always reliable for picking up genetic conditions that are caused by repeat expansions. Additionally, many people think that because NIPT has a high level of accuracy, it is capable of diagnosing conditions. However, diagnostic tests like amniocentesis or chorionic villus sampling are still needed if someone screens positive with NIPT, because if the genetic condition is rare, there are still a lot of false-positive results.

Multiple key challenges impact NGS for clinical implementation and utility:

1. Tissue is the issue: A small biopsy or degraded sample may not yield sufficient tumor DNA or RNA.
2. Tumor heterogeneity: A single biopsy may not capture the full mutational landscape, leading to sampling bias.
3. Variation of panel design, reporting and interpretation.
4. Data complexity in the bioinformatics: NGS requires advanced technical and computational infrastructure and skilled personnel to handle testing, data processing, storage interpretation and data integration.
5. Long turnaround times: This test is complex and involves multiple steps, requiring multiple members of the pathology team with various expertise. The estimated turenaround time for in-house testing is 10 business days, whole send-out testing may take longer due to the additional time required for shipping the tissue.
6. High initial and operational [costs]: Because of equipment, reagents and personnel.
7. Insurance coverage for NGS-based tests may be limited or variable depending on indication in the location.
8. Detection of incidental findings, for example, germline mutations, that may raise ethical dilemmas.
9. Appropriate informed consent and the genetic counseling framework must be considered.

While constantly emerging new cancer variants and biomarkers require frequent updates to panels pending interpretation pipelines, regulatory and accreditation requirements add complexity to clinical implementation.

While physicians acknowledge that NGS testing has the potential to match patients with cutting-edge targeted therapies that can significantly improve outcomes, its use in routine clinical practice remains limited due to these challenges.