A new version of NGS-QC-PANEL enables better authentication and characterization of human and mouse samples

Abstract Introduction: We previously reported a deep NGS-based QC panel for the authentication, characterization, and contamination of human and mouse samples [1]. It has visibly higher sensitivity and accuracy than traditional STR and SNP assays in authenticating human and mouse cancer cell lines, patient-derived xenograft tumors, and organoids, and is able to detect both inter- and intra-species contamination. Using NGS data from a single run, it also provides other functionalities, including the detection of mycoplasma contamination and viral infection of HBV/EBV/HPV16/HPV18/HIV viruses; the identification of gender and genetic background; the estimation of genetic drift and the construction of phylogenetic trees for samples with a common ancestor; and the quantification of percentages for individual cell lines in their mixtures. A new version of the NGS-QC-PANEL was developed to further enhance the application of the current version. Methods: The new version has over 2000 pairs of primers, compared to 600 pairs in the previous version. We designed characteristic SNPs for more mouse models and selected the characteristic segments for specific mycoplasma species, more microorganism species, and some vertebrate species. Also, we selected more segments for gender characterization. The primers were designed for these newly added SNPs and segments and incorporated into the previous primer pools. Results: The number of mouse models that can be unambiguously authenticated increases from 32 to 92, in particular, MC38 of 5 different origins can be distinguished. Besides quantifying mycoplasma contamination by calculating a ratio of mycoplasma cells to human/mouse cells, the panel is able to distinguish 85 mycoplasma species. More than 100 virus and bacteria species can be detected. Three more segments, in addition to the existing three, are added to infer the gender of human models. Finally, the panel can detect cross-species contamination from cat, cattle, chicken, dog, duck, guineapig, hamster, monkey, rabbit, rat, sheep based on species-specific segments. Conclusions: The new NGS-QC-PANEL makes the authentication, characterization, and contamination detection for human and mouse samples more accurate, more comprehensive and more in-depth, which will further contribute to quality control and assurance of the preclinical tumor models used in drug discovery. References: [1]Chen, X., Qian, W., Song, Z., Li, Q. X., & Guo, S. (2020). Authentication, characterization and contamination detection of cell lines, xenografts and organoids by barcode deep NGS sequencing. NAR Genomics and Bioinformatics, 2(3), lqaa060. Citation Format: Wubin Qian, Xiaobo Chen, Sheng Guo, Wubin Qian, Yanghui Sheng. A new version of NGS-QC-PANEL enables better authentication and characterization of human and mouse samples [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A009.