Challenges of Next-Generation–Sequencing-Based Molecular Diagnostics

Abstract

Although targeted sequencing by next-generation sequencing (NGS) technology has gained much attention in the field of molecular diagnostics, several limitations have delayed its application to medical genetics. It is difficult to standardize the procedures because of the use of various sequencing platforms and target enrichment methods, which are updated rapidly. The cutoff thresholds for accurate variant identification, including the minimum read depth, range of variant percentage compared with the wild type, and quality score, have not been fully defined. Variable coverage depth across target regions and misalignment between homologous sequences or pseudogenes may also affect the accuracy of sequencing data. The fast falling sequencing cost and rapid development of NGS analyzing bioinformatics pipeline make it possible to use gene panels, whole exome sequencing (WES), and whole genome sequencing (WGS) for clinical diagnoses. However, there are many challenges in developing and implementing NGS technology for clinical use. Understanding these challenges can help molecular diagnostic professionals and ordering health professionals better gauge the utility of NGS clinical tests and the interpretation of the NGS results. NGS is a complex sequencing technology and many challenging steps need to be carried out to complete the test to generate clinically useful interpretations.