Abstract 738: Highly multiplexed and precisely calibrated reference materials for copy number variation detection

Abstract

Abstract Introduction: Genomic instability is a hallmark of cancer, and copy number variation (CNV), including gene duplication and deletion, can be a key driver of oncogenesis. Detection of CNV is needed for personalized treatment, and may involve microarray analysis or Next Generation Sequencing. Often, DNA from patient-derived cell lines is used as positive control material in these assays. However, these materials are often poorly characterized, may change over time, and have additional confounding genetic alterations. Here we describe the development of new reference materials that are highly characterized by digital PCR and can aid in the validation and calibration of new diagnostic assays for CNV. Methods: The GM24385 reference cell line has been extensively characterized by the Genome in a Bottle project1 and originates from a participant in the Personal Genome Project. DNA from this cell line was used to make a library of full-length gene inserts. The library had >12X coverage of the GM24385 genome and an average insert size of ~135 Kb. PCR based screening was used to identify and isolate full length MYC, FGFR3 and ERBB2 genes. Next Generation Sequencing, using Illumina Nextera library preparation kits and a MiSeq instrument, was used to verify the gene of interest. The genomic DNA and gene inserts were then formulated into reference materials, in purified DNA format to amplify the copy numbers of these key genes. Digital PCR was used to control the mixing and verify the number of copies of genes and/or mutations within the reference material. Results: The sequence confirmed genes were formulated into GM24385 genomic DNA at levels consistent with 5X and 10X amplification. Testing on the ArcherDx VariantPlex™ Solid Tumor Kit indicated that each amplification could be detected at the expected level. Conclusions: Highly characterized and consistently manufactured reference materials for copy number detection are currently lacking. The described Seraseq CNV Reference Materials provide proof of concept for a biosynthetic, engineered material that uses digital PCR as an orthologous reference method and provides a “ground truth” copy number level for key oncogenes. Citation Format: Catherine Huang, Yves Konigshofer, Jessica Dickens, Bharathi Anekella. Highly multiplexed and precisely calibrated reference materials for copy number variation detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 738. doi:10.1158/1538-7445.AM2017-738