Next generation sequencing and the future of genetic diagnosis

Abstract

The introduction of next generation sequencing (NGS) has led to an exponential increase of elucidated genetic causes in both extremely rare diseases and common but heterogeneous disorders. It can be applied to the whole or to selected parts of the genome (genome or exome sequencing, gene panels). NGS is not only useful in large extended families with linkage information, but may also be applied to detect de novo mutations or mosaicism in sporadic patients without a prior hypothesis about the mutated gene. Currently, NGS is applied in both research and clinical settings, and there is a rapid transition of research findings to diagnostic applications. These developments may greatly help to minimize the "diagnostic odyssey" for patients as whole-genome analysis can be performed in a few days at reasonable costs compared with gene-by-gene analysis based on Sanger sequencing following diverse clinical tests. Despite the enthusiasm about NGS, one has to keep in mind its limitations, such as a coverage and accuracy of < 100%, resulting in missing variants and false positive findings. In addition, variant interpretation is challenging as there is usually more than one candidate variant found. Therefore, there is an urgent need to define standards for NGS with respect to run quality and variant interpretation, as well as mechanisms of quality control. Further, there are ethical challenges including incidental findings and how to guide unaffected probands seeking direct-to-customer testing. However, taken together, the application of NGS in research and diagnostics provides a tremendous opportunity to better serve our patients.

Fig. 1

Workflow of a next generation sequencing (NGS) analysis. The figure provides a simplified overview of the main phases in NGS and demonstrates some of the different options at each step. For the sequencing analysis, DNA is required that can be derived from large families with many affected, sporadic patients, and their healthy parents (trios), or several small families with the same disease (for gene discovery) or from individual patients (for diagnostic purposes). In the second step, DNA needs to be prepared for the sequencing by fragmentation if the whole genome is the target for NGS. Alternatively, specific target sequences need to be enriched (for instance, by hybridization or polymerase chain reaction). Third, the equally sized fragments have to be ligated to universal adapters, clonally amplified, and loaded onto a chip. Next, the chip is placed into an NGS machine and the sequencing reaction (integration of a nucleotide) is monitored by a light signal (fluorescence) or by the release of a proton resulting in a pH change. While all of these first steps are carried out at the bench in a genetic laboratory (“wet lab”), the next 3 steps are performed at a desk using a computer and several software packages and databases (“dry lab”). Specifically, the recorded signal from the NGS machine needs to be translated into a sequence, which is aligned to the reference genome. Next, mismatches with the reference sequence are retrieved and annotated with respect to the coding part of the genome. This is followed by the critical step of variant interpretation involving the separation of likely benign from possibly pathogenic variants. For this, information is collected and evaluated for categorization of the effect on an encoded protein, for in silico prediction of the consequences on protein function, and for previously reported knowledge on the gene and the specific mutation in question from databases. Finally, when one or a few candidate variants are selected as potential disease cause, comprehensive validation is needed and takes place in the “wet lab”. Validation must include resequencing to separate false positive from true positive variants, screening of ethnically matched controls, tests for the effect of the mutation on protein function, or studies of the mutated protein in animal models