Validation of a next-generation sequencing oncology panel optimized for low input DNA

Area of Science:

• Oncology
• Genomics
• Molecular Diagnostics

Background:

• Next-generation sequencing (NGS) in clinical oncology requires substantial DNA input, limiting its application in challenging samples.
• Developing low-input DNA assays is crucial for broader clinical utility.

Purpose of the Study:

• To develop and validate a focused NGS panel (Penn Precision Panel, PPP) for detecting clinically significant variants in cancer.
• To optimize the panel for minimal DNA input (0.5-10 ng) from solid tumor specimens.

Main Methods:

• Designed a targeted NGS panel (PPP) covering 20 cancer-relevant genes.
• Analyzed 123 samples, including 83 FFPE solid tumors, using 0.5-10 ng DNA input.
• Sequenced libraries on an Illumina MiSeq platform with high read depth (>6500x).
• Utilized an in-house pipeline for variant detection and analysis.

Main Results:

• Achieved high clinical sensitivity (96.75%) and specificity (99.9%) for variant detection.
• Demonstrated strong concordance in variant allele frequencies (VAFs) with a validated panel (r=0.98).
• Successfully analyzed low-yield solid tumor specimens.

Conclusions:

• The Penn Precision Panel (PPP) is a robust and clinically validated NGS test for low-input DNA.
• PPP effectively captures clinically relevant variants, comparable to larger panels, with significantly reduced DNA requirements.
• This assay expands the utility of NGS in clinical oncology, especially for limited sample quantities.