Validation and implementation of next-generation sequencing technologies in a clinical molecular diagnostic laboratory

Next-generation sequencing (NGS) technologies representpowerful tools capable of massive parallel sequencing of DNA. Their application has revolutionized characterization of genomic aberrations responsible for initiation and maintenance of cancers, resulting in a high discovery rate of therapeutic and prognosticmarkers. In a clinical molecular diagnosticlaboratory, the high sequencing capacitiesof NGS technologies are well suited for routinescreening of increasing number of markers using low inputs of DNA in high sample volumes. However, implementation of these technologies in the clinical environment needsthorough validation of their workflow suitability, their capability to detect a varietyof genomic aberrations, and their efficiencyin comparison to other orthogonal sequencing platforms being routinely used. Here, using a targeted NGS panel to screen for mutational hot spots in 46 cancer-relatedgenes as an example, we have discussed various parameters which need to be establishedfor validation and implementation of the NGS assays. We have highlighted various assayperformance metrics which need to be established toward complete validation of the NGSplatform before implementation. The criteria for filtering, annotating, and clinical reporting of variants are also discussed.