Diagnostic applications of next generation sequencing: working towards quality standards/Diagnostische Anwendung von Next Generation Sequencing: Auf dem Weg zu Qualitätsstandards

Abstract

Over the past 6 years, next generation sequencing (NGS) has been established as a valuable high-throughput method for research in molecular genetics and has successfully been employed in the identifi cation of rare and common genetic variations. All major NGS technology companies providing commercially available instruments (Roche 454, Illumina, Life Technologies) have recently marketed bench top sequencing instruments with lower throughput and shorter run times, thereby broadening the applications of NGS and opening the technology to the potential use for clinical diagnostics. Although the high expectations regarding the discovery of new diagnostic targets and an overall reduction of cost have been achieved, technological challenges in instrument handling, robustness of the chemistry and data analysis need to be overcome. To facilitate the implementation of NGS as a routine method in molecular diagnostics, consistent quality standards need to be developed. Here the authors give an overview of the current standards in protocols and workfl ows and discuss possible approaches to defi ne quality criteria for NGS in molecular genetic diagnostics.Keywords: bioinformatics; genetic variation; Illumina; library preparation; Life Technologies; molecular genetics; molecular genetic diagnostics; next generation sequencing (NGS); rare diseases; Roche. ZusammenfassungIn den vergangenen 6 Jahren hat sich " next generation sequencing " (NGS) als wichtige Hochdurchsatz-Methode f ü r die molekulargenetische Forschung etabliert und wurde erfolgreich zur Identifi kation seltener und h ä ufi ger genetischer Varianten eingesetzt. Alle gr ö ß eren NGS-Technologieunternehmen, die bisher kommerziell erh ä ltliche Ger ä te zu Verf ü gung stellten (Roche 454, Illumina, Life Technologies), haben vor kurzem auch "benchtop " -Ger ä te mit geringerem Durchsatz und k ü rzeren Laufzeiten auf den Markt gebracht, wodurch die Anwendungsgebiete erweitert und der Technologie der m ö gliche Einsatz in der klinischen Diagnostik er ö ffnet wurde. W ä hrend die hohen Erwartungen hinsichtlich der Entdeckung neuer diagnostischer Zielstrukturen und einer Senkung der Kosten erreicht wurden, m ü ssen etliche technologische Herausforderungen hinsichtlich Bedienung der Ger ä te, Robustheit der Chemie und Handhabung der Datenanalyse noch gemeistert werden. Um die Einf ü hrung von NGS in die Routinediagnostik zu erleichtern, m ü ssen nachhaltige Qualit ä tsstandards entwickelt werden. Im Folgenden geben die Autoren einen Ü berblick ü ber die gegenw ä rtigen Standards in den Protokollen und Arbeitsabl ä ufen und diskutieren m ö gliche Ans ä tze Qualit ä tskriterien f ü r NGS in der molekulargenetischen Diagnostik zu defi nieren.

Dealing with the incidental finding of secondary variants by the example of SRNS patients undergoing targeted next-generation sequencing

“…A growing number of enrichment methods and platforms are now available for specific applications from targeted resequencing to wholegenome sequencing based on massively parallel sequencing of short DNA fragments [45]. Therefore, with the particular devices, the application range was considerably broadened and a transition into the field of clinical diagnostics was opened [1]. All platforms have in common that they perform sequencing of millions of small fragments of DNA in parallel.…”

“…Furthermore, NGS allows the study of larger regions of the genome or even the whole genome, an approach that would be too cost-, labor-, and time-intensive with conventional Sanger sequencing. The application of NGS techniques has therewith the potential to broaden the diagnostic spectrum from Mendelian diseases to polygenic disorders [1]. We established targeted-NGS in our laboratory for mutational analysis in patients with steroid-resistant nephrotic syndrome (SRNS).…”

Quality assurance and standardization in view of non-invasive prenatal testing (NIPT)

“…For the implementation of NGS in diagnostics, the need for quality standards was noted years ago and guidelines have already been published [25,26]. For a better explanation of the quality needs, the analysis with NGS in the given context will be summarized and split into a technical phase and a statistical/bioinformatics phase.…”

“…For a better explanation of the quality needs, the analysis with NGS in the given context will be summarized and split into a technical phase and a statistical/bioinformatics phase. Detailed descriptions of NGS workflows are available [25,27].…”

German Guidelines for Molecular Genetic Diagnostic Testing Using High-throughput Technology, Such As Next-Generation Sequencing

“…NGS technology has only recently been transferred to patient care applications, and its longevity remains unclear in the light of emerging technologies, such as single-molecule sequencing, which are already under evaluation [1]. In recent years, the clinical application of NGS, in particular genepanel sequencing, has been evaluated rigorously in parallel to addressing target enrichment [2], quality-related workflow elements [3], and the bioinformatics pipeline [4]. Clinical whole genome sequencing (cWGS) is now technically possible and eagerly anticipated as a high-quality clinical diagnostic tool; however, this technique has not yet captured much of the diagnostic market.…”