Targeted Next-Generation Sequencing: The Clinician's Stethoscope for Genetic Disorders

Jan Haas; Ioana Barb; Hugo A Katus; Benjamin Meder


Personalized Medicine. 2014;11(6):581-592. 

In This Article


Over the last decade, NGS has evolved into an extremely valuable tool, which has triggered a paradigm shift in the field of human genetics. Researchers and physicians have been provided with an exceptional instrument for deciphering the human genome in a fast and increasingly cost-efficient way. However, the big challenge we are facing is to make best use of this new technology in the clinical setting. One of the main problems associated with the clinical implementation of NGS is the lack of a proper infrastructure, which does not only refer to practical or technical issues, such as data production, processing and storage, but also to medical and regulatory aspects. With the FDA approval of the Illumina MiSeqDx, one important hurdle for the translation of NGS into the clinical arena has been overcome. As this system has a limited throughput, it especially qualifies for sequencing subgenomes relevant for specific diseases. In this context, target enrichment methods are best to answer most clinical question in cases where a known set of genes needs to be investigated. During test development, the stages from sample enrichment, sequencing and data analysis must be optimized and standard operation procedures need to be developed.[10,30] Optimization steps should aim to improve sample pooling, capture efficiency and coverage maximization. In parallel, data analysis needs to be adapted to the specific technology and workflow and benchmarked to reference samples.[72] Finally, clinical tests must undergo continuous quality control cycles to keep highest standards over time.

While there is a multitude of targeted NGS approaches, none of them is ideal or suitable for every application, because each has its unique advantages and disadvantages.[3] As previously discussed, all methods have demonstrated to principally produce good results, more or less worthy of a clinical diagnostic test. However, important aspects such as target size, number of analyzed samples, laboratory equipment and budget, processing time, the utilized sequencing instrument and ultimately the required performance in terms of specificity, sensitivity and uniformity need to be carefully considered in order to choose the most suitable targeted approach for each application. In addition to the generation of high-quality variants, also standards for interpretation of variants in a clinical context and the presentation of results to the physician and patient must be established. Here, a reasonable categorization of variants ranges from 'disease causing' and 'likely disease causing' to 'variant of unknown clinical significance'.[30]