Table 1:

Summary of circulating tumor DNA detection techniques.

Technique	Example technologies	Scale of analysis	Method	Detection limit (% of Cost cfDNA)		Assay personalization	Advantages	Limitations
AS-PCR	ARMS [112]	Single-locus or multiplexed assays	Preferentially amplifies rare mutant DNA molecules	~ 0.1–1	$	Some required	Ease of use; ideal for detecting recurrent ‘hotspot’ point mutations	Can only query small number of variants concurrently; cannot detect mutations not known a priori
dPCR	dPCR [117] ddPCR [119,125,126,127] BEAMing [128, 129]	Single-locus or multiplexed assays	Partitions target DNA into different reactions for massively parallel qPCR	~0.01	$$	Some required	High sensitivity	Can only query small number of variants concurrently; cannot detect mutations not known a priori
WGS	WGS [143, 144] Plasma-Seq [139, 140] PARE [145]	Genome-wide	NGS of whole genome	~10	$$– $$$	Not required	Entire genome is interrogated	Low sensitivity; mostly limited to SCNA detection
	FAST-SeqS [141]
Retrotransposon-based amplicon NGS	mFAST-SeqS [152] WALDO [142]	Genome-wide retrotransposon sites	PCR amplification of retrotransposon insertion sites prior to NGS analysis	~ 5	$$	Not required	Rapid aneuploidy assessment with lower cost than WGS	Limited to aneuploidy detection
WES	WES [79]	Exome-wide	NGS of whole exome	~ 5	$$$	Not required	Entire exome is interrogated	Low sensitivity
Multiplex PCR-based NGS	TAm-Seq [155] Enhanced TAm-Seq [156] Safe-SeqS [111] Natera® [64]	Targeted sequencing	PCR amplification enriches targets prior to NGS analysis	~ 0.01–2.0	$$	Some required	High sensitivity (modern methods)	Less comprehensive than other NGS methods; unable to detect SCNAs; unable to detect fusions without assay personalization
Hybrid Capture-based NGS	CAPP-Seq [70,84] TEC-Seq [73] Guardant360® [161, 162] FoundationOne® Liquid [163]	Targeted sequencing	Subset of exome is hybridized to biotinylated probes and captured for NGS analysis	~ 0.001–0.5	$$	Not required	High sensitivity; detects multiple mutation types; broadly applicable without personalization	Less comprehensive than WGS or WES
Combination approaches	CAPP-Seq + GRP [149] CancerSEEK [169] UroSEEK [7]	Single-locus to genome-wide	Combines different ctDNA detection methods, sometimes including protein biomarkers	Variable	$$–$$$	Variable	Improved detection compared to standard ctDNA analysis alone in certain settings	Potentially more time and resource intensive

ARMS Amplification Refractory Mutation System, AS-PCR allele-specific PCR, 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 PCR, dPCR digital PCR, FAST-SeqS Fast Aneuploidy Screening Test-Sequencing System, GRP Genome Representation Profiling, mFAST-SeqS modified FAST-SeqS, NGS next-generation sequencing, PARE Personalized Analysis of Rearranged Ends, Plasma-Seq plasma sequencing, Safe-SeqS Safe Sequencing System, SCNA somatic copy number alternation, TAm-Seq Tagged-Amplicon Sequencing, TEC-Seq Targeted Error Correction Sequencing, WALDO Within Sample Aneuploidy Detection, WES whole exome sequencing, WGS whole genome sequencing