BACKGROUND: Targeted sequencing using oncopanels requires comprehensive assessments of accuracy and detection sensitivity to ensure analytical validity. By employing reference materials characterized by the U.S. Food and Drug Administration-led SEquence Quality Control project phase2 (SEQC2) effort, we perform a cross-platform multi-lab evaluation of eight Pan-Cancer panels to assess best practices for oncopanel sequencing. RESULTS: All panels demonstrate high sensitivity across targeted high-confidence coding regions and variant types for the variants previously verified to have variant allele frequency (VAF) in the 5-20% range. Sensitivity is reduced by utilizing VAF thresholds due to inherent variability in VAF measurements. Enforcing a VAF threshold for reporting has a positive impact on reducing false positive calls. Importantly, the false positive rate is found to be significantly higher outside the high-confidence coding regions, resulting in lower reproducibility. Thus, region restriction and VAF thresholds lead to low relative technical variability in estimating promising biomarkers and tumor mutational burden. CONCLUSION: This comprehensive study provides actionable guidelines for oncopanel sequencing and clear evidence that supports a simplified approach to assess the analytical performance of oncopanels. It will facilitate the rapid implementation, validation, and quality control of oncopanels in clinical use.

• Klíčová slova
• Analytical performance, Molecular diagnostics, Oncopanel sequencing, Precision medicine, Reproducibility, Target enrichment,
• MeSH
• diagnostické techniky molekulární metody   normy   MeSH
• genetické testování metody   normy   MeSH
• genomika metody   normy   MeSH
• jednonukleotidový polymorfismus MeSH
• lidé MeSH
• mutace MeSH
• nádorové biomarkery * MeSH
• nádory diagnóza   genetika   MeSH
• onkogeny * MeSH
• reprodukovatelnost výsledků MeSH
• senzitivita a specificita MeSH
• variabilita počtu kopií segmentů DNA MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Intramural MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• nádorové biomarkery * MeSH

BACKGROUND: Oncopanel genomic testing, which identifies important somatic variants, is increasingly common in medical practice and especially in clinical trials. Currently, there is a paucity of reliable genomic reference samples having a suitably large number of pre-identified variants for properly assessing oncopanel assay analytical quality and performance. The FDA-led Sequencing and Quality Control Phase 2 (SEQC2) consortium analyze ten diverse cancer cell lines individually and their pool, termed Sample A, to develop a reference sample with suitably large numbers of coding positions with known (variant) positives and negatives for properly evaluating oncopanel analytical performance. RESULTS: In reference Sample A, we identify more than 40,000 variants down to 1% allele frequency with more than 25,000 variants having less than 20% allele frequency with 1653 variants in COSMIC-related genes. This is 5-100× more than existing commercially available samples. We also identify an unprecedented number of negative positions in coding regions, allowing statistical rigor in assessing limit-of-detection, sensitivity, and precision. Over 300 loci are randomly selected and independently verified via droplet digital PCR with 100% concordance. Agilent normal reference Sample B can be admixed with Sample A to create new samples with a similar number of known variants at much lower allele frequency than what exists in Sample A natively, including known variants having allele frequency of 0.02%, a range suitable for assessing liquid biopsy panels. CONCLUSION: These new reference samples and their admixtures provide superior capability for performing oncopanel quality control, analytical accuracy, and validation for small to large oncopanels and liquid biopsy assays.

• MeSH
• alely * MeSH
• frekvence genu * MeSH
• genetická heterogenita MeSH
• genetická variace * MeSH
• genetické testování metody   normy   MeSH
• genomika metody   normy   MeSH
• lidé MeSH
• nádorové biomarkery * MeSH
• nádorové buněčné linie MeSH
• nádory diagnóza   genetika   MeSH
• průběh práce MeSH
• variabilita počtu kopií segmentů DNA MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• nádorové biomarkery * MeSH