Table 2.

Advantages and limitations of reviewed techniques in FH diagnosis

	Advantages	Limitations
Sanger sequencing	• Time-efficient and cost-effective for low numbers of targets • Single base-pair resolution • 5% minor allele fractions can be detected • Contiguous sequences as long as 1000 bases can be analyzed • Useful in NGS confirmation	• Not cost-effective for high target numbers • Low scalability due to increasing sample input requirements
NGS	• Sequencing multiple genes/ targets simultaneously • High sample throughput • Single base-pair resolution • Higher probability to find novel variants • Increased sequencing depth resulting in higher sensitivity (down to 1%)	• Less cost-effective and less time-efficient for sequencing low target numbers (up to 20) • Big data interpretation is needed
Single nucleotide polymorphism genotyping	• Cost-effective for SNPs • Time-efficient results • Can be scaled up in multiplex SNP assays	• Interrogating one SNP at a time • Investigates point mutations and not DNA aberrations
Biochip array technology	• ICD certified (based on the British population) • The mutational status can be determined from a single test	• Fixed number of investigated mutations/SNPs • Limited published data available
Multiplex ligation-dependent probe amplification	• Golden standard technique for CNV (microdeletions, microduplications) identification • P-062 investigates 33 LDLR targets in one reaction • IVD certified • Multiplex, easy workflow, and low-cost technique	• Investigates only the LDLR gene • Unable to detect anomalies at the single cell level • Cannot detect unknown point mutations • Sensitive to novel/undescribed benign polymorphisms at or near a probe ligation site