Design and analytical validation of clinical DNA sequencing assays

Context.-DNA sequencing is the method of choice for mutation detection in many genes. Objectives.-To demonstrate the analytical accuracy and reliability of DNA sequencing assays developed in clinical laboratories. Only general guidelines exist for the validation of these tests. We provide examples of assay validation strategies for DNA sequencing tests. Design.-We discuss important design and validation considerations. Results.-The validation examples include an accuracy study to evaluate concordance between results obtained by the newly designed assay and analyzed by another method or laboratory. Precision (reproducibility) studies are performed to determine the robustness of the assay. To assess the quality of sequencing assays, several sequence quality measures are available. In addition, assessing the ability of primers to specifically and robustly amplify target regions before sequencing is important. Conclusion.-Protocols for validation of laboratorydeveloped sequencing assays may vary between laboratories. An example summary of a validation is provided.