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Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies
Published on: April 11, 2016
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Inter-assay variability of next-generation sequencing-based gene panels.
Pham Nguyen Quy1, Keita Fukuyama1,2, Masashi Kanai3
1Department of Therapeutic Oncology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
BMC Medical Genomics
|April 16, 2022
Summary
Preclinical factors like sample type and gene panel design significantly impact next-generation sequencing (NGS) assay concordance. FFPE samples and tumor-only panels showed higher discordance rates, affecting clinical variant interpretation.
Area of Science:
- Genomics and Molecular Diagnostics
- Oncology
- Clinical Laboratory Science
Background:
- Tumor heterogeneity is a known cause of inter-assay discordance in next-generation sequencing (NGS).
- The influence of preclinical factors, including sample type, quality, and gene panel analytical features, on NGS concordance remains understudied.
Purpose of the Study:
- To investigate how sample type (FFPE vs. FF) and gene panel analytical features affect concordance between different NGS assays.
- To quantify inter-assay discordance rates in clinical settings.
Main Methods:
- DNA samples from formalin-fixed paraffin-embedded (FFPE) and fresh-frozen (FF) tissues were analyzed using tumor-only (TO) and paired tumor-normal (TN) gene panels.
- Replicate FFPE samples from the same block were tested with two independent TN panel assays.
- Variants reported by TO and TN panels were compared, and discordance rates were calculated.
Main Results:
- A substantial overall discordance rate of 71.8% was observed between the two NGS panels, with significantly higher rates for FFPE samples (p < 0.05).
- Among variants unique to the TO panel, 32.3% were germline variants excluded by the TN panel, and 30.3% had low allele frequencies, potentially indicating artificial calls.
- Independent TN panel assays on the same FFPE block also yielded a high discordance rate of 55.3%.
Conclusions:
- Inter-NGS assay discordance in clinical settings is frequently driven by sample type and variations in panel analytical features.
- These findings highlight the critical need to consider sample characteristics and assay design when interpreting NGS results for clinical decision-making.
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