When should next‑generation sequencing (NGS) be used for genetic testing?

When to Use Next-Generation Sequencing (NGS) for Genetic Testing

NGS should be used for genetic testing when multiple genes need to be evaluated simultaneously, particularly in metastatic cancers with actionable mutations, inherited disorders with genetic heterogeneity, and when traditional single-gene testing would be inefficient or deplete limited tissue samples.

Cancer/Oncology Applications

Strongly Recommended Indications

For lung adenocarcinoma, NGS is recommended to assess level I alterations (EGFR, ALK, ROS1, BRAF V600E, MET exon 14 skipping) as these directly guide FDA-approved targeted therapies. 1 The ESMO guidelines emphasize that NGS can be performed on either RNA or DNA, provided the panel includes level I fusions. This approach is superior to serial single-gene testing because it:

• Prevents tissue depletion from multiple separate tests
• Reduces diagnostic delays
• Identifies multiple actionable targets simultaneously 2

For prostate cancer and cholangiocarcinoma, multigene NGS is recommended to detect level I alterations, with larger panels acceptable only if payers cover the full strategy cost and panels accurately rank alterations by clinical actionability. 1

For carcinoma of unknown primary (CUP), large panel NGS is recommended as it helps identify the tissue of origin and detects actionable mutations, with TMB-high status associated with 60% response rates to immunotherapy versus 7.7% in TMB-low patients. 3

Conditional Recommendations

For ovarian cancer, NGS can determine somatic BRCA1/2 mutations for PARP inhibitor eligibility. 1

For colon cancer, NGS is an alternative to PCR only if it doesn't increase costs. 1

No current indication exists for NGS in squamous cell lung cancer, breast cancer, gastric cancer, pancreatic cancer, or hepatocellular carcinoma from a public health perspective, though clinical research centers should perform multigene sequencing in molecular screening programs. 1

Inherited/Genetic Disorders

Cholestatic Liver Disease

NGS techniques such as panel sequencing and whole-exome sequencing (WES) are recommended early in the diagnostic work-up for cholestatic liver disease without clear cause in infants and children. 4 In adults, test after excluding common causes, particularly with unusual features or treatment resistance.

Critical point: Sanger sequencing of individual genes is now discouraged except possibly for ABCB4 testing in young patients with small duct primary sclerosing cholangitis. 4 Since multiple genes can present with similar phenotypes, panel approaches are superior.

Living Kidney Donor Evaluation

When a potential living donor has a positive family history of kidney disease (at least one family member with known/suspected genetic kidney disease or unknown cause of early-onset kidney disease), NGS should be used to identify the familial variant. 5 The algorithm is:

1. Test the affected family member (usually recipient) first with disease-focused or comprehensive genetic testing
2. If diagnosis confirmed, test donor for the specific familial variant
3. Positive result in donor → counsel against donation
4. Negative result → permit donation with counseling about residual uncertainty

Rare Genetic Disorders with Heterogeneity

NGS is cost-effective for rapid diagnosis of rare disorders with genetic heterogeneity, including:

• Glycogen storage diseases
• Familial intrahepatic cholestasis
• Lysosomal storage diseases
• Primary immunodeficiencies 6

These conditions would otherwise require sequential single-gene testing, which is time-consuming and expensive.

Technical Considerations

Panel Selection

The choice between targeted panels, WES, and WGS depends on the clinical scenario:

• Targeted panels: Best for known gene sets with established clinical utility, offering better coverage depth, faster turnaround, lower cost, and simpler data handling 7, 8
• WES: Appropriate when the causative gene is unknown but likely in coding regions; allows reanalysis as new disease genes are discovered 4, 6
• WGS: Consider only when WES fails to identify causative mutations 4

Critical Limitations to Recognize

NGS panels vary significantly in what they detect - not all panels identify fusions, copy number variations, or structural variants. 2, 3 Clinicians must verify that the specific panel includes detection methods for the alterations relevant to their patient's condition.

For fusion gene detection specifically: RNA-based sequencing is superior to DNA-based methods because it identifies the actual fusion transcript and its functionality, whereas DNA sequencing may miss breakpoints in large intronic regions. 3

Reanalysis is essential: For patients without diagnosis after initial testing, reanalyze data at least every 3 years as new disease genes and variants are discovered. 4

When NOT to Use NGS

Do not use NGS when:

• A single well-characterized gene explains the phenotype and Sanger sequencing is sufficient (rare exception)
• Standard biochemical tests can establish diagnosis without genetic confirmation
• The patient's condition has no established genetic basis
• Results would not change management and patient/family are not seeking reproductive counseling

Quality Requirements

Any NGS test used clinically must:

• Detect actionable mutations occurring with ≥1% frequency 2
• Include appropriate quality control and validation
• Provide accurate variant ranking by clinical actionability 1
• Be interpreted by teams including bioinformaticians, clinicians, and laboratory staff in collaboration 8

Genetic counseling must precede testing, with discussion of incidental findings, variants of uncertain significance (VUS), and limitations of interpretation. 4