Validations of variants captured in custom canine cancer panels using the Sanger method
Cancer is a disease in which cells in the body acquire ways to grow uncontrollably and lose their capacity to function normally. This causes a chain reaction that negatively impacts day to day function and may eventually lead to death if left untreated. Pet dogs provide a unique opportunity to understand the genetic components of human cancers. Not only do domestic dogs have similar genes to humans, they also share similar environments and are afflicted with similar cancers. Furthermore, canine cancer progression is found to run the same course as human cancer. All these factors contribute to pet dogs being a good model organism to study human cancers. Finally, studying canine cancers will help further cancer diagnosis and treatments for our pet dogs.
Previously at Vidium Animal Health, a next generation sequencing (NGS) based canine cancer panel was designed using hybrid-capture technology. This oncopanel, called SearchLight DNA™, searches for variants in genes commonly mutated in cancers. SearchLight DNA™ was designed to be applicable for many types of canine cancers, such as melanoma, hemangiosarcoma and lung cancers. This panel contains 120 genes that encompasses canine equivalent genes found in a human based cancer panel, canine cancer associated hotspots and genes known to be amplified or deleted in cancers. These variants are known predictive, diagnostic and prognostic biomarkers.
Though NGS-based oncopanels are becoming more widely accepted in the medical community, there is still a need to validate mutations by using Sanger sequencing. Sanger is considered the “gold standard” of DNA sequencing due to its 99.99% accuracy. We have recently sequenced 2 canine hemangiosarcoma cell lines using the SearchLight DNA™ assay. NGS data was analyzed and seven relevant variants were chosen for validation with Sanger sequencing. Primers for these specific variants were designed using Primer3 software and the PCR products were sent to Genewiz for Sanger validation. The Sanger data revealed that of the 7 variants, 2 variants were confirmed whereas the other 5 variants either failed at the PCR level or at the Sanger sequencing step due to non-specificity. Further work is needed to validate these last five variants and also other variants reported by the NGS panel.