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.

Future Perspective

Over the next years, we expect the clinical use of NGS to expand considerably. The outstanding improvement in throughput and the concomitant drop in costs are rapidly driving NGS from the research scene into the clinical arena. We expect target enrichment NGS to become a routine clinical diagnostic tool for a multitude of inherited diseases. For genetically more complex diseases, the implementation of whole-exome and whole-genome sequencing will require further efforts to overcome challenges in bioinformatics infrastructure and analysis methods. Despite all challenges and unanswered questions, it is hard not to be optimistic about the future of personalized genetic sequencing and its prospective impact on patient care management.

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Financial & competing interests disclosure
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.

Abstract and Introduction
Background
Translation of NGS Into the Clinics
Benchmarking of Selective Circularization Enrichment Against Hybridization
Conclusion
Future Perspective
References
Sidebar

Table 1. Currently available enrichment technologies.
Target enrichment method	Advantages	Disadvantages	Target region size	Library preparation time	Complexity	Special equipment	Relative costs	Lowest DNA input required	Sample pooling
Selective circularization	Ideal for medium target sizes (100–500 kb) High specificity Time-effective No dedicated instrument/equipment needed	Capture deficiency across repetitive or GC-rich regions Uneven coverage	1 kb to 5 Mb	1–2 days	Medium	None	Kits	200 ng	Postcapture
Hybridization	Ideal for larger target regions (500 kb whole exome) High multiplexing capacity High scalability No dedicated instrument/equipment needed	Capture deficiency across repetitive or GC-rich regions Time-consuming library preparation	<50 Mb	3–5 days	High	None	Kits	200 ng	Precapture/postcapture
Multiplex PCR	Ideal for smaller targets (10–100 kb) Fast and easy workflows High specificity Effective also for low-quality DNA No dedicated instrument/equipment needed Low amount of input DNA	Sequence variation in the primer binding site might lead to amplicon loss	<5 Mb	<1 day	Medium	None	Kits	10 ng per primer pool	After PCR, before library preparation
Microdroplet PCR	Automation	Instrument needed	20,000 genomic loci per sample	<1 day	Low	Instrument	Kits and instrument	250 ng	Postenrichment
PCR (microfluidic chip)	Automation	Instrument needed	48 samples with 480 amplicons	<1 day	Low	Instrument	Kits and instrument	50 ng	Postenrichment