Functional DNA quantification guides accurate next-generation sequencing mutation detection in formalin-fixed,...

Area of Science:

• Molecular biology
• Genomics
• Biotechnology

Background:

• Formalin-fixed, paraffin-embedded (FFPE) tissues are standard clinical specimens.
• Molecular assays like targeted next-generation sequencing (NGS) face challenges with FFPE DNA quality.
• Accurate DNA quantification is essential for reliable molecular testing of FFPE samples.

Purpose of the Study:

• To compare different DNA quantification methods for FFPE samples.
• To introduce and evaluate a novel quantitative PCR (QFI-PCR) assay for FFPE DNA.
• To demonstrate the impact of improved FFPE DNA quality assessment on NGS accuracy.

Main Methods:

• Comparison of three DNA quantification methods, including a novel quantitative PCR (QFI-PCR) assay.
• Analysis of 165 residual clinical FFPE specimens.
• Evaluation of the impact on targeted next-generation sequencing (NGS) mutation detection.

Main Results:

• Commonly used FFPE DNA quantification methods have limitations.
• The QFI-PCR assay accurately measures amplifiable DNA copy number.
• An integrated workflow using QFI-PCR enhances NGS accuracy and rescues low-quality FFPE DNA.

Conclusions:

• QFI-PCR provides a valuable tool for assessing FFPE DNA quality.
• Improved quality control measures are needed for NGS-based patient testing.
• This approach can optimize FFPE DNA input for more accurate molecular diagnostics.