BACKGROUND: Clinical laboratories routinely use formalin-fixed paraffin-embedded (FFPE) tissue or cell block cytology samples in oncology panel sequencing to identify mutations that can predict patient response to targeted therapy. To understand the technical error due to FFPE processing, a robustly characterized diploid cell line was used to create FFPE samples with four different pre-tissue processing formalin fixation times. A total of 96 FFPE sections were then distributed to different laboratories for targeted sequencing analysis by four oncopanels, and variants resulting from technical error were identified. RESULTS: Tissue sections that fail more frequently show low cellularity, lower than recommended library preparation DNA input, or target sequencing depth. Importantly, sections from block surfaces are more likely to show FFPE-specific errors, akin to "edge effects" seen in histology, while the inner samples display no quality degradation related to fixation time. CONCLUSIONS: To assure reliable results, we recommend avoiding the block surface portion and restricting mutation detection to genomic regions of high confidence.
In recent years, an emerging dermocystidiosis caused by Dermocystidium anguillae Spangenberg, 1975 has been found to pose a threat to the culture of American eel, Anguilla rostrata (Lesueur), as well as Chinese perch, Siniperca chuatsi (Basilewsky), in China. Dermocystidium anguillae was originally described from European eel, Anguilla anguilla (Linnaeus), and it is thus important to identify the possible source of this pathogen. In the present study, we compared D. anguillae from European eels cultured in China with those from American eels. Molecular analysis showed that the SSU rDNA of D. anguillae infecting European eels was identical to that of D. anguillae infecting American eels, suggesting their conspecificity. To investigate the source of D. anguillae causing dermocystidiosis in American eels cultured in China, a specific PCR assay for the detection of D. anguillae was developed with high sensitivity (10-6 ng/μl of D. anguillae genomic DNA). Using the present molecular detection method, the water and sediment of culture ponds, fish feed and American eel elvers imported from America were screened for the presence of D. anguillae. No amplicons were detected from the water, sediment and fish feed samples. However, positive amplicons were found in American eel elvers, indicating that D. anguillae has been introduced from American eel elvers to China. It is suggested that American eel elvers imported from America should be examined for the presence of D. anguillae before their exportation abroad to prevent the spread of this pathogen.
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
Circulating tumor DNA (ctDNA) sequencing is being rapidly adopted in precision oncology, but the accuracy, sensitivity and reproducibility of ctDNA assays is poorly understood. Here we report the findings of a multi-site, cross-platform evaluation of the analytical performance of five industry-leading ctDNA assays. We evaluated each stage of the ctDNA sequencing workflow with simulations, synthetic DNA spike-in experiments and proficiency testing on standardized, cell-line-derived reference samples. Above 0.5% variant allele frequency, ctDNA mutations were detected with high sensitivity, precision and reproducibility by all five assays, whereas, below this limit, detection became unreliable and varied widely between assays, especially when input material was limited. Missed mutations (false negatives) were more common than erroneous candidates (false positives), indicating that the reliable sampling of rare ctDNA fragments is the key challenge for ctDNA assays. This comprehensive evaluation of the analytical performance of ctDNA assays serves to inform best practice guidelines and provides a resource for precision oncology.
- MeSH
- cirkulující nádorová DNA genetika MeSH
- individualizovaná medicína * MeSH
- lékařská onkologie * MeSH
- lidé MeSH
- limita detekce MeSH
- nádory genetika MeSH
- reprodukovatelnost výsledků MeSH
- sekvenční analýza DNA normy MeSH
- směrnice pro lékařskou praxi jako téma MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, N.I.H., Intramural MeSH
- validační studie MeSH
