Potential pitfalls in multiplex PCR-based next-generation sequencing: a case-based report

Area of Science:

• Oncology
• Genetics
• Molecular Diagnostics

Background:

• Amplicon-based next-generation sequencing (NGS) is a sensitive, rapid, and cost-effective method for detecting clinically actionable mutations.
• Accurate detection of these mutations is vital for cancer diagnosis, prognosis, and treatment selection.
• However, inherent limitations in amplicon-based NGS assays can impact variant interpretation.

Purpose of the Study:

• To highlight potential pitfalls associated with amplicon-based NGS assays.
• To provide insights based on institutional experience.
• To offer strategies for minimizing risks in clinical variant reporting.

Main Methods:

• Review of institutional experience with amplicon-based NGS assays.
• Identification and illustration of specific assay limitations.
• Development of recommendations for risk mitigation.

Main Results:

• Three distinct potential pitfalls in amplicon-based NGS assays were identified.
• These pitfalls can affect the accuracy of variant detection and interpretation.
• Strategies to minimize these risks were proposed.

Conclusions:

• Awareness of amplicon-based NGS assay limitations is essential for correct clinical variant interpretation.
• Proactive measures can mitigate risks associated with these assays.
• Implementing these strategies enhances the reliability of NGS in cancer patient management.