Universal Reference RNA as a standard for microarray experiments

BACKGROUND: Obtaining reliable and reproducible two-color microarray gene expression data is critically important for understanding the biological significance of perturbations made on a cellular system. Microarray design, RNA preparation and labeling, hybridization conditions and data acquisition and analysis are variables difficult to simultaneously control. A useful tool for monitoring and controlling intra- and inter-experimental variation is Universal Reference RNA (URR), developed with the goal of providing hybridization signal at each microarray probe location (spot). Measuring signal at each spot as the ratio of experimental RNA to reference RNA targets, rather than relying on absolute signal intensity, decreases variability by normalizing signal output in any two-color hybridization experiment. RESULTS: Human, mouse and rat URR (UHRR, UMRR and URRR, respectively) were prepared from pools of RNA derived from individual cell lines representing different tissues. A variety of microarrays were used to determine percentage of spots hybridizing with URR and producing signal above a user defined threshold (microarray coverage). Microarray coverage was consistently greater than 80% for all arrays tested. We confirmed that individual cell lines contribute their own unique set of genes to URR, arguing for a pool of RNA from several cell lines as a better configuration for URR as opposed to a single cell line source for URR. Microarray coverage comparing two separately prepared batches each of UHRR, UMRR and URRR were highly correlated (Pearson's correlation coefficients of 0.97). CONCLUSION: Results of this study demonstrate that large quantities of pooled RNA from individual cell lines are reproducibly prepared and possess diverse gene representation. This type of reference provides a standard for reducing variation in microarray experiments and allows more reliable comparison of gene expression data within and between experiments and laboratories

DNA-based Fish Species Identification Protocol

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. This versatile method employs PCR amplification of genomic DNA extracted from fish samples, followed by restriction fragment length polymorphism (RFLP) analysis to generate fragment patterns that can be resolved on the Agilent 2100 Bioanalyzer and matched to the correct species using RFLP pattern matching software

Cross-oncopanel study reveals high sensitivity and accuracy with overall analytical performance depending on genomic regions.

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. 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. 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.All SEQC2 participants freely donated their time, reagents, and computing resources for the completion and analysis of this project. Part of this work was carried out with the support of the Intramural Research Program of the National Institutes of Health (to Mehdi Pirooznia), National Institute of Environmental Health Sciences (to Pierre Bushel), and National Library of Medicine (to Danielle Thierry-Mieg, Jean Thierry-Mieg, and Chunlin Xiao). Leming Shi and Yuanting Zheng were supported by the National Key R&D Project of China (2018YFE0201600), the National Natural Science Foundation of China (31720103909), and Shanghai Municipal Science and Technology Major Project (2017SHZDZX01). Donald J. Johann, Jr. acknowledges the support by FDA BAA grant HHSF223201510172C. Timothy Mercer and Ira Deveson were supported by the National Health and Medical Research Council (NHMRC) of Australia grants APP1108254, APP1114016, and APP1173594 and Cancer Institute NSW Early Career Fellowship 2018/ECF013. This research has also been, in part, financially supported by the MEYS of the CR under the project CEITEC 2020 (LQ1601), by MH CR, grant No. (NV19-03-00091). Part of this work was carried out with the support of research infrastructure EATRIS-CZ, ID number LM2015064, funded by MEYS CR. Boris Tichy and Nikola Tom were supported by research infrastructure EATRIS-CZ, ID number LM2018133 funded by MEYS CR and MEYS CR project CEITEC 2020 (LQ1601).S

A verified genomic reference sample for assessing performance of cancer panels detecting small variants of low allele frequency

none74siBackground 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.noneJones, Wendell; Gong, Binsheng; Novoradovskaya, Natalia; Li, Dan; Kusko, Rebecca; Richmond, Todd A.; Johann, Donald J.; Bisgin, Halil; Sahraeian, Sayed Mohammad Ebrahim; Bushel, Pierre R.; Pirooznia, Mehdi; Wilkins, Katherine; Chierici, Marco; Bao, Wenjun; Basehore, Lee Scott; Lucas, Anne Bergstrom; Burgess, Daniel; Butler, Daniel J.; Cawley, Simon; Chang, Chia-Jung; Chen, Guangchun; Chen, Tao; Chen, Yun-Ching; Craig, Daniel J.; del Pozo, Angela; Foox, Jonathan; Francescatto, Margherita; Fu, Yutao; Furlanello, Cesare; Giorda, Kristina; Grist, Kira P.; Guan, Meijian; Hao, Yingyi; Happe, Scott; Hariani, Gunjan; Haseley, Nathan; Jasper, Jeff; Jurman, Giuseppe; Kreil, David Philip; Łabaj, Paweł; Lai, Kevin; Li, Jianying; Li, Quan-Zhen; Li, Yulong; Li, Zhiguang; Liu, Zhichao; López, Mario Solís; Miclaus, Kelci; Miller, Raymond; Mittal, Vinay K.; Mohiyuddin, Marghoob; Pabón-Peña, Carlos; Parsons, Barbara L.; Qiu, Fujun; Scherer, Andreas; Shi, Tieliu; Stiegelmeyer, Suzy; Suo, Chen; Tom, Nikola; Wang, Dong; Wen, Zhining; Wu, Leihong; Xiao, Wenzhong; Xu, Chang; Yu, Ying; Zhang, Jiyang; Zhang, Yifan; Zhang, Zhihong; Zheng, Yuanting; Mason, Christopher E.; Willey, James C.; Tong, Weida; Shi, Leming; Xu, JoshuaJones, Wendell; Gong, Binsheng; Novoradovskaya, Natalia; Li, Dan; Kusko, Rebecca; Richmond, Todd A.; Johann, Donald J.; Bisgin, Halil; Sahraeian, Sayed Mohammad Ebrahim; Bushel, Pierre R.; Pirooznia, Mehdi; Wilkins, Katherine; Chierici, Marco; Bao, Wenjun; Basehore, Lee Scott; Lucas, Anne Bergstrom; Burgess, Daniel; Butler, Daniel J.; Cawley, Simon; Chang, Chia-Jung; Chen, Guangchun; Chen, Tao; Chen, Yun-Ching; Craig, Daniel J.; del Pozo, Angela; Foox, Jonathan; Francescatto, Margherita; Fu, Yutao; Furlanello, Cesare; Giorda, Kristina; Grist, Kira P.; Guan, Meijian; Hao, Yingyi; Happe, Scott; Hariani, Gunjan; Haseley, Nathan; Jasper, Jeff; Jurman, Giuseppe; Kreil, David Philip; Łabaj, Paweł; Lai, Kevin; Li, Jianying; Li, Quan-Zhen; Li, Yulong; Li, Zhiguang; Liu, Zhichao; López, Mario Solís; Miclaus, Kelci; Miller, Raymond; Mittal, Vinay K.; Mohiyuddin, Marghoob; Pabón-Peña, Carlos; Parsons, Barbara L.; Qiu, Fujun; Scherer, Andreas; Shi, Tieliu; Stiegelmeyer, Suzy; Suo, Chen; Tom, Nikola; Wang, Dong; Wen, Zhining; Wu, Leihong; Xiao, Wenzhong; Xu, Chang; Yu, Ying; Zhang, Jiyang; Zhang, Yifan; Zhang, Zhihong; Zheng, Yuanting; Mason, Christopher E.; Willey, James C.; Tong, Weida; Shi, Leming; Xu, Joshu