Table 1.

Analytical methods for plasma cfDNA genotyping

Assay category	Assay technology/name (regulatory approval)	Analysis scale	Method	Limit of detection (as % of cfDNA)	Advantages
Quantitative PCR	qPCR	Single mutations or panels of known and well‐characterized mutations	Preferentially amplifies rare mutant DNA molecules	~0.1%–1%	Highly sensitive and specific. Low turnaround time.
	cobas EGFR Mutation Test v2 (FDA and EMA approved)
	therascreen EGFR Plasma RGQ PCR kit
	PANAMutyper R EGFR kit
Digital PCR	ddPCR	Single‐locus or multiplexed assays	Partitions target DNA into different reactions for massively parallel qPCR	~0.04%–0.1%	Highly sensitive and specific.
	Bio‐Rad QX200 ddPCR Dx system
	BEAMing
	OncoBEAM EGFR kit
NGS whole genome and exome sequencing	Whole genome sequencing	Genome wide Exome wide	NGS of whole genome or whole exome	~10% for whole genome ~5% for whole exome	Evaluation of entire genome or exome can lead to discovery of new targets. Exome sequencing allows rapid aneuploidy assessment with lower cost than whole genome sequencing. Discovery of mechanisms of resistance.
	Roche/454
	Ion Torrent: Proton/PGM
	Illumina Sequencing (Solexa)
	SOLiD
Hybrid capture‐based NGS	Targeted NGS sequencing	Targeted sequencing of captured regions of the genome	Subset of exome is hybridized to biotinylated probes and captured for NGS analysis	~0.001%–0.5%	Highly sensitive. Simultaneous detection of predetermined genes of interest. Comprehensive detection of known and unknown mutations. Lower cost and less bioinformatics data compared with whole genome sequencing.
	Guardant360 CDx (FDA approved)
	FoundationOne Liquid CDx (FDA approved)
	Resolution ctDX Lung
	CAPP‐Seq
	TEC‐Seq
Multiplex PCR‐based NGS	Targeted NGS sequencing	Targeted sequencing of predefined regions	PCR amplification enriches targets before NGS analysis	~0.01–2.0%	Highly sensitive.
	TAm‐Seq
	Enhanced TAm‐Seq
	Safe‐SeqS
	Natera
Combination approaches (including DNA + biomarkers)	CAPP‐Seq + GRP	Single locus to genome wide	Combines different ctDNA detection methods, sometimes including protein biomarkers	Variable	Improved detection compared with standard ctDNA analysis alone in certain settings.
	CancerSEEK
	UroSEEK

Abbreviations: BEAMing, beads, emulsion, amplification and magnetics; CAPP‐Seq, cancer personalized profiling by deep sequencing; cfDNA, cell‐free DNA; ctDNA, circulating tumor DNA; ddPCR, digital droplet polymerase chain reaction; EGFR, epidermal growth factor receptor; EMA, European Medicines Agency; FDA, U.S. Food and Drug Administration; NGS, next‐generation sequencing; PGM, personal genome machine; PIK3CA, phosphatidylinositol‐4,5‐bisphosphate 3‐kinase catalytic subunit alpha; qPCR, quantitative polymerase chain reaction; Safe‐SeqS, Safe‐Sequencing System; SOLiD, sequencing by oligonucleotide ligation and detection; TAm‐Seq, tagged‐amplicon deep sequencing.