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.

Abstract and Introduction

Abstract

Genetic biomarkers are crucial for diagnosis, guiding of treatments and estimation of prognosis. In the past, clinical genetic diagnostics was limited by the sequencing information gained from selected exons and single genes. For genetically heterogeneous diseases, such as cardiomyopathies, where underlying mutations in more than 1000 exons are known, a Sanger-based comprehensive test would have been extremely expensive and labor intensive. Next-generation sequencing has overcome these problems in terms of costs, speed and throughput. In this review we discuss available methods for targeted next-generation sequencing that ease the introduction of this technology into routine clinical application. We further provide results of a study we have performed to compare two state-of-the-art methods for their enrichment efficiency and detection accuracy of variants in a clinical setting.

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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