Abstract
The application of next-generation sequencing (NGS) to characterize cancer genomes has resulted in the discovery of numerous genetic markers. Consequently, the number of markers that warrant routine screening in molecular diagnostic laboratories, often from limited tumor material, has increased. This increased demand has been difficult to manage by traditional low- and/or medium-throughput sequencing platforms. Massively parallel sequencing capabilities of NGS provide a much-needed alternative for mutation screening in multiple genes with a single low investment of DNA. However, implementation of NGS technologies, most of which are for research use only (RUO), in a diagnostic laboratory, needs extensive validation in order to establish Clinical Laboratory Improvement Amendments (CLIA) and College of American Pathologists (CAP)-compliant performance characteristics. Here, we have reviewed approaches for validation of NGS technology for routine screening of tumors. We discuss the criteria for selecting gene markers to include in the NGS panel and the deciding factors for selecting target capture approaches and sequencing platforms. We also discuss challenges in result reporting, storage and retrieval of the voluminous sequencing data and the future potential of clinical NGS.
Highlights
Characterizing genomic aberrations in tumors for predictive and prognostic purposes by genome sequencing has become an integral part of the precision medicine approach [1,2,3,4,5,6]
Most of the techniques used for this purpose included low- and medium-throughput traditional techniques such as Sanger sequencing, pyrosequencing, allele-specific polymerase chain reaction (PCR), high-resolution melt curve analysis, fragment analysis, and primer-extension coupled with mass spectroscopy
The increased discovery rate of clinically relevant biomarkers as a result of extensive characterization of cancer genomes has necessitated the testing of multiple genes per tumor as the standard-of-care
Summary
Characterizing genomic aberrations in tumors for predictive and prognostic purposes by genome sequencing has become an integral part of the precision medicine approach [1,2,3,4,5,6]. Compared with earlier genome characterization techniques, NGS technologies possess distinct advantages more suitable to addressing the challenges associated with the increasing demands for testing of multiple gene markers with lower inputs of nucleic acids. Screening multiple markers with NGS technology requires a single input of relatively low-quantity DNA or RNA in contrast to traditional sequencing technologies, which need cumulatively larger quantities of input nucleic acid. NGS is quantitative as the allelic fraction of the mutation can be gleaned by the number of DNA strands with mutation in the background of strands with the wild-type sequence These considerable advantages make NGS very desirable for routine clinical sequencing of tumors; implementation of NGS poses several challenges, which we discuss below
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