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 laboratory-developed sequencing assays may vary between laboratories. An example summary of a validation is provided.

[1]  W. Grody,et al.  ACMG recommendations for standards for interpretation and reporting of sequence variations: Revisions 2007 , 2008, Genetics in Medicine.

[2]  Rong Mao,et al.  Mutation database for the galactose‐1‐phosphate uridyltransferase (GALT) gene , 2007, Human mutation.

[3]  Weimin Sun,et al.  Extensive sequencing of the cystic fibrosis transmembrane regulator gene: Assay validation and unexpected benefits of developing a comprehensive test , 2003, Genetics in Medicine.

[4]  P. Green,et al.  Base-calling of automated sequencer traces using phred. I. Accuracy assessment. , 1998, Genome research.

[5]  P Green,et al.  Base-calling of automated sequencer traces using phred. II. Error probabilities. , 1998, Genome research.

[6]  Kathleen M Murphy,et al.  Comparison of Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations: diagnostic and clinical implications. , 2010, The Journal of molecular diagnostics : JMD.

[7]  S Rozen,et al.  Primer3 on the WWW for general users and for biologist programmers. , 2000, Methods in molecular biology.

[8]  Association for Molecular Pathology statement. Recommendations for in-house development and operation of molecular diagnostic tests. , 1999, American journal of clinical pathology.

[9]  M. Gulley,et al.  Clinical laboratory reports in molecular pathology. , 2007, Archives of pathology & laboratory medicine.

[10]  Kelli Sumner,et al.  The Alport syndrome COL4A5 variant database , 2010, Human mutation.