Table 2.

Comparison of targeted PCR-based assays and NGS for DNA analysis (adopted from [9,12,22]).

	Method	Limit of Detection	Advantages	Disadvantages	Clinical Use
Targeted PCR-Based Assays
Digital PCR (ddPCR, BEAMing)	DNA fractionation into different reactions sites for parallel qPCR	0.04–0.1%	Highly sensitive and specific Quantitative Low turnaround time	Not suitable for unknown alterations	Resistance genotyping
qPCR (e.g., Cobas®, Therascreen®)	Amplification of predefined DNA sequences	0.1–1%	Highly sensitive and specific Low turnaround time	Limited multiplexing Semi-quantitative	Initial and resistance genotyping of known mutations
Next generation sequencing (NGS) methods
WGS	NGS of the full genome	10%	Detection of unknown alterations and new mechanisms of resistance (MOR)	Low specificity (false positives) Risk of detecting germline mutations Low sensitivity extensive bioinformatics High costs	Not in clinical routine, more experimental use
WES	NGS of the full exome (i.e., coding regions)	5%
Hybrid-capture based NGS (e.g., Guardant360® CDx, FoundationOne® Liquid CDx)	Sequencing of target regions, that are captured by hybridization	0.001–0.5%	High sensitivity detection of SNPs, CNVs and gene fusions Simultaneous detection of predefined genes of interest as well as unknown mutations	Lower specificity (65%) than amplicon-based NGS unable to detect fusions without prior knowledge of partners	Initial and resistance genotyping
Amplicon-based (PCR-capture) NGS	Sequencing of target regions, that are amplified by PCR	0.01–2%	High sensitivity detection of SNPs, CNVs and gene fusions Simultaneous detection of predefined genes of interest as well as unknown mutations Higher specificity than hybrid-capture NGS (>99%)	Bias of CNVs and AFs (due to amplification) Unable to detect fusions without prior knowledge of partners