Review of Clinical Next-Generation Sequencing

Abstract

Context.-Next-generation sequencing (NGS) is a technology being used by many laboratories to test for inherited disorders and tumor mutations. This technology is new for many practicing pathologists, who may not be familiar with the uses, methodology, and limitations of NGS.Objective.-To familiarize pathologists with several aspects of NGS, including current and expanding uses; methodology including wet bench aspects, bioinformatics, and interpretation; validation and proficiency; limitations; and issues related to the integration of NGS data into patient care.Data Sources.-The review is based on peer-reviewed literature and personal experience using NGS in a clinical setting at a major academic center.Conclusions.-The clinical applications of NGS will increase as the technology, bioinformatics, and resources evolve to address the limitations and improve quality of results. The challenge for clinical laboratories is to ensure testing is clinically relevant, cost-effective, and can be integrated into clinical care.( As with any new technology, the use of NGS in the clinical laboratory has evolved and will continue to evolve over time. New applications for the technology continue to be developed, new bioinformatics and wet bench techniques are being developed to address current limitations and improve performance, and new knowledge regarding interpretation of rare variants is being accumulated. This article is an overview of clinical NGS, including recent trends as well as evolution that will likely occur in the near future. The review is based on peer-reviewed literature and personal experience using NGS in a clinical setting at a major academic center. The Molecular Diagnostics Laboratory at

Molecular genetic testing methodologies in hematopoietic diseases: current and future methods

“…While all methods have caveats and advantages to be aware of, it is also important to be aware that the data analysis pipeline is critical in any NGS platform. Both customized commercially available and in‐house analysis pipelines are commonly used . Indeed the performance of an NGS assay depends not only on the physical assay and instrumentation itself, but the computational/analytical tools being used or which have been developed.…”

“…Both customized commercially available and in-house analysis pipelines are commonly used. [58][59][60] Indeed the performance of an NGS assay depends not only on the physical assay and instrumentation itself, but the computational/analytical tools being used or which have been developed. As such, it is critical to know the weaknesses and issues with any NGS workflow.…”

A novel, homozygous nonsense variant of the CDHR1 gene in a Chinese family causes autosomal recessive retinal dystrophy by NGS‐based genetic diagnosis

“…Here, we applied targeted next‐generation sequencing (TGS) technology, the most available ‎and promising method available, to identify a novel, homologous mutation of CDHR1 gene in a Chinese family with autosomal recessive retinal dystrophy, extending the gene's mutation spectrum.…”

“…CDHR1 is a photoreceptor-specific cadherin, 9 and immunoprecipitation studies showed that CDHR1 are also found in complexes with PROM1 and actin filaments, playing a critical role in photoreceptor disk morphogenesis. 10 Here, we applied targeted next-generation sequencing (TGS) technology, the most available and promising method available, [11][12][13][14][15][16] to identify a novel, homologous mutation of CDHR1 gene in a Chinese family with autosomal recessive retinal dystrophy, extending the gene's mutation spectrum.…”

Single Genes, Panels, and Next-Generation Sequencing Platforms: A Financial Perspective

“…If the laboratory does not already perform NGS for other indications, this investment can be on the order of hundreds of thousands of dollars. 1 More ''turnkey'' solutions that include bioinformatics processing, variant annotation, and storage solutions may mitigate the investments required in these areas, but they may be limited in capability and cost more. Single biomarker tests, by contrast, performed with more traditional methodologies (eg, Sanger sequencing), require less capital investment, as the infrastructure and platforms generally exist in the laboratory.…”