Quality metrics for enhanced performance of an NGS panel using single-vial amplification technology

Area of Science:

• Molecular Oncology
• Genomic Medicine
• Biotechnology

Background:

• Targeted next-generation sequencing (NGS) panels are crucial in molecular oncology.
• Low DNA quantity and quality from formalin-fixed paraffin-embedded tissues and paucicellular specimens pose challenges for NGS.
• Accurate genomic profiling is essential for precision cancer therapy.

Purpose of the Study:

• To describe a novel NGS assay utilizing single tube stem-loop inhibition mediated amplification technology.
• To demonstrate the assay's capability to deliver highly accurate results with low DNA input.
• To validate the assay for clinical laboratory use with rigorous quality metrics.

Main Methods:

• A customized NGS panel targeting 48 genes in solid tumors was employed.
• Validation involved sequencing patient samples with 136 known variants (SNVs, indels) across various specimen types.
• Specimens included FFPE blocks, core biopsies, and cytology material with neoplastic cellularity ranging from 10% to 80%.

Main Results:

• The assay demonstrated high specificity, sensitivity, accuracy, reproducibility, and precision.
• Concordant results were achieved with as little as 2-3 ng DNA input, 10% neoplastic cellularity, and variant allelic frequencies of 2.5%-3%.
• Over 99% of target areas achieved ≥500X coverage, with a 95.3% success rate in reporting results from over 2000 clinical specimens.

Conclusions:

• The advanced NGS technology enables accurate identification of clinically relevant genomic variants.
• The assay offers uniform coverage, impressive turnaround time, and a cost-effective workflow.
• This validated test is suitable for routine clinical molecular oncology laboratories.