Background: Application of next generation sequencing (NGS) is revolutionizing the clinical molecular diagnostics industry. As this occurs, guidelines for validating NGS processes are limited, non-specific, and rapidly evolving. NGS validation projects are complex and expensive, so the validation experiments must be carefully considered, while being certain all current and evolving regulatory requirements are met.
Methods: We validated an end-to-end genetic testing process for a 16-gene hereditary colorectal cancer NGS panel. Global work flow was designed to provide high quality sequencing data for 100% of our targeted regions. Assay parameters tested were accuracy, precision, reportable range, reference range, analytical sensitivity, and analytical specificity.
Results: Five instrument runs were conducted, encompassing 115 samples, through an end-to-end process validation. Overall accuracy of the NGS portion of the assay was 99.98%. Single nucleotide variant detection accuracy was 100%. Insertion/deletion variant (INDEL) detection accuracy was 100% for INDELs of one to eight nucleotides and 97% overall (range one to 14 nucleotides) by NGS. There were no false negatives within our data set for a sensitivity of 100%.
Conclusion: Given the relative novelty of NGS in the clinical setting and rapidly evolving external guidance, clinical laboratories must develop their own strategies to design, develop, validate, and implement NGS assays. Our experiences highlight a chosen strategy as well as some of the benefits and limitations of NGS.