Progress and prospects of next-generation sequencing testing for inherited retinal dystrophy

Figures & data

Table 1. Comparison of molecular diagnostic strategies for inherited retinal dystrophies.

Approach	Method		Advantages	Disadvantages	Detects
					Novel variants	Novel genes
Hypothesis driven	SSCP		Low cost	Small DNA fragments only; optimal fragment size 150–200bp. Requires confirmation by Sanger. Sensitivity (mutation detection rate) less than Sanger sequencing, at ∼80–90%	✓	✗
	Microarray		Low cost. Detects mutations across multiple genes	Only known mutations can be identified	✗	✗
	Sanger sequencing		Well-established method for clinical testing	High cost; cannot be scaled up. Large/heterozygous insertion/deletion mutations cannot be detected (but smaller homozygous insertion/deletion mutations can be detected). Mutations outside the sequenced regions cannot be detected	✓	✗
	Targeted NGS	Disease-specific panel	Low cost; massively paralleled sequencing of selected genes simultaneously	Less cost-effective when clinical diagnosis is not certain. Typically longer turnaround time. Detection of insertion/deletion mutations can be a challenge. Mutations outside the sequenced regions cannot be detected. Repetitive regions may not be covered well	✓	✗
Non-hypothesis driven	Panel NGS	Standard RD panel	Broader target region than disease-specific panels. More specific than WES; offering higher and typically better coverage of the genes of interest	Detection of insertion/deletion mutations can be a challenge. Mutations outside the targeted regions cannot be detected. Repetitive regions may not be covered well. Coverage varies	✓	✗
		Smart RD panel	Unmatched specificity by simplex PCR; a simple and fast method of target enrichment	Insertion/deletion mutations cannot be detected. Mutations outside the sequenced regions cannot be detected. Repetitive regions may not be covered well	✓	✗
	Whole exome NGS	WES	An acceptable choice when clinical diagnosis is uncertain. Lower cost than ordering multiple panels	Coverage is uneven. Incidental findings can create ethical dilemmas. Detection of insertion/deletion mutations can be a challenge. Mutations outside the captured regions cannot be detected. Repetitive regions may not be covered well	✓	✓
	Whole genome NGS	WGS	No target enrichment bias	Repetitive regions may not be covered well. Data interpretation is a major bottleneck. Years away from truly affordable clinical testing. Incidental findings can create ethical dilemmas; should the entire data be returned back to patients? Data delivery and storage issues	✓	✓

NGS: Next-generation sequencing; SSCP: Single strand conformation polymorphism; WES: Whole exome sequencing; WGS: Whole genome sequencing.