Background and Objectives: Accurate screening of clinically significant tumor mutations is critical for precision medicine in oncology. This requires genotyping platforms with high accuracy and compatibility with varying DNA yields from challenging sample types. Here, we have validated a new, improved, compact, and self-contained pyrosequencing platform (Pyromark Q48 Autoprep; Q48) for screening N-, K- and H-RAS mutations in thyroid cancers. Methods: A set of 73 thyroid cancer and 16 non-thyroid cancer samples (fine needle aspirates and formalin-fixed paraffin-embedded) with known mutation status of RAS genes were tested using the Q48 platform. Performance parameters such as accuracy, precision, and limit-of-detection were established. Q48 workflow was compared to an older Q96 pyrosequencing platform to highlight the differences and advantages. RAS testing by pyrosequencing was also compared to a clinically validated next-generation sequencing platform using 56 thyroid cancer samples. Results: The Q48 Pyromark was found to be a very reliable platform suited for quick testing of RAS genes with complete accuracy, high precision, and high detection sensitivity. It had comparable accuracy, with higher sequencing success rates than NGS. The hands-on time, workflow ease, and efficiency were also significantly improved in comparison with the Q96 platform. Conclusions: Overall, the Q48 platform was found to be a well-suited and agile clinical sequencing platform to rapidly screen RAS mutations.
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