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Updated: Apr 21, 2026
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
Evaluation of an integrated clinical workflow for targeted next-generation sequencing of low-quality tumor DNA using
Background:
Improvements in both performance and cost for next-generation sequencing (NGS) have spurred its rapid adoption for clinical applications. We designed and optimized a pan-cancer target-enrichment panel for 51 well-established oncogenes and tumor suppressors, in conjunction with a bioinformatic pipeline informed by in-process controls and pre- and post-analytical quality control measures.
Methods:
The evaluation of this workflow consisted of sequencing mixtures of intact DNA to establish analytical sensitivity and precision, utilization of heuristics to identify systematic artifacts, titration studies of intact and FFPE samples for input optimization, and incorporation of orthogonal sequencing strategies to increase both positive predictive value and variant detection. We also used 128 FFPE samples to assess clinical accuracy and incorporated the previously described quantitative functional index (QFI) for sample qualification as part of detailing complete system performance.
Results:
We observed a concordance correlation coefficient of 0.99 between the observed versus expected percent variant at 250 ng input across 4 independent sequencing runs. A subset of the systematic variants were confirmed to be barely detectable on an independent sequencing platform (Wilcox signed-rank test p-value <10(-16)), and the incorporation of orthogonal sequencing strategies increased the harmonic mean of sensitivity and positive predictive value of mutation detection by 41%. In one cohort of FFPE tumor samples, coverage and inter-platform concordance were positively correlated with the QFI, emphasizing the need for pre-analytical sample quality control to reduce the risk of false positives and negatives. In a separate cohort of FFPE samples, the 51-gene panel achieved 78% sensitivity (95% CI = 56.3, 92.5) with 100% PPV (95% CI = 81.5, 100.0) based on known mutations at 7.9% median abundance. By sequencing specimens using an orthogonal NGS technology, sensitivity was improved to 87.0% (95% CI = 66.4,97.2) while maintaining PPV.
Conclusions:
The results highlight the value of process integration in a comprehensive targeted NGS system, enabling both discovery and diagnostic applications, particularly when sequencing low-quality cancer specimens.
Insights
This study optimized a targeted next-generation sequencing (NGS) panel for cancer genes, improving accuracy and sensitivity for clinical diagnostics, especially with challenging FFPE samples.
Area of Science:
- Genomics
- Oncology
- Molecular Diagnostics
Background:
- Next-generation sequencing (NGS) adoption in clinical settings is increasing due to performance and cost improvements.
- A pan-cancer target-enrichment panel for 51 oncogenes and tumor suppressors was developed.
- A comprehensive bioinformatic pipeline with quality control measures was integrated.
Purpose of the Study:
- To design and optimize a targeted NGS workflow for clinical cancer gene analysis.
- To evaluate the analytical sensitivity, precision, and clinical accuracy of the developed panel and pipeline.
- To assess the impact of pre-analytical sample quality on diagnostic performance.
Main Methods:
- Sequencing of DNA mixtures to determine analytical sensitivity and precision.
- Heuristics for artifact identification and titration studies for input optimization.
- Orthogonal sequencing strategies and quantitative functional index (QFI) for performance enhancement and sample qualification.
- Clinical accuracy assessment using 128 FFPE samples.
Main Results:
- High concordance (CCC=0.99) between observed and expected variant percentages.
- Orthogonal sequencing increased mutation detection sensitivity and positive predictive value by 41%.
- The 51-gene panel achieved 78% sensitivity and 100% PPV in FFPE samples, improving to 87% sensitivity with orthogonal sequencing.
Conclusions:
- Process integration in targeted NGS systems enhances both discovery and diagnostic applications.
- The optimized workflow demonstrates value in sequencing low-quality cancer specimens.
- Emphasis on pre-analytical sample quality control is crucial for reducing false positives and negatives.
